Early Pregnancy Factor Research Articles

Early Pregnancy Factor and Implantation on Domestic Animals

Early Pregnancy Factor and Implantation on Domestic Animals

Mare's Early Pregnancy Factor (EPF): Study of purification method

Mare's Early Pregnancy Factor (EPF): Study of purification method

Early pregnancy factor: an unresolved molecule.

Early pregnancy factor: an unresolved molecule.

An update on the identity of early pregnancy factor and its role in early pregnancy.

An update on the identity of early pregnancy factor and its role in early pregnancy.

An update on the identity of early pregnancy factor and its role in early pregnancy.

An update on the identity of early pregnancy factor and its role in early pregnancy.

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.

Detection of Bovine Early Pregnancy Factor in the Culture Medium of Sperm-Injected or Plasmamembrane-Perforated Oocytes.

This study was undertaken to determine the mechanism of early pregnancy factor (EPF) secretion from ova. EPF released into the culture medium from in vitro matured bovine oocytes that were sperm-injected and plasmamembrane-perforated was detected. Bovine follicular oocytes were matured in vitro for 19 to 22 h in 5% CO2 in air at 39 C. A capacitated spermatozoa was injected into each of the three parts of the matured oocyte, namely, the zona pellucida, the perivitelline space and the ooplasm. The plasmamembrane of matured oocytes were also perforated with a glass micropipette into the ooplasm. After sperm-injection or perforation, these ova were cultured separately in TCM199 containing 2% fetal calf serum (FCS) for 24 h in 5% CO2 in air at 39 C. Then EPF activity in these culture media were assayed by rosette inhibition test. A rosette inhibition titer (RIT) above 4 was considered as a positive expression of EPF activity. Of the medium samples from the sperm-injected oocytes, 4 out of 5 of those injected in the perivitelline space and 4 out of 5 of those injected in the ooplasm showed positive EPF activity. Further, all 7 medium samples of oocytes perforated in the plasmamembrane with a glass micropipette also showed positive EPF activity. However, all 4 medium samples containing ova injected with sperm into the zona pellucida showed negative EPF activity. Consideration of these data led to a remarkable discription of EPF release from oocytes micromanipulated with sperm and inorganic matter, and provided the evidence to show that EPF release is triggered by sperm penetrating through the zona pellucida.

Isolation and Purification of Early Pregnancy Factor (EPF) in Rat Serum.

Early pregnancy factor (EPF) has been detected in pregnancy sera of many animal species. Isolation and purification of the EPF in pregnancy rat serum (Day 4) was attempted by using ammonium sulfate fractionation, ion-exchange chromatography, affinity chromatography and Immunoblotting. As a result, rat EPF was detected in 40% ammonium sulfate supernatant, CM-Sepharose Fast Flow 20 mM and 30 mM NaCl elution fraction, DEAE-Sepharose Fast Flow unbound fraction and Affinity chromatography unbound fraction. By the result of immunoblotting, there were three bands (22 KD, 24 KD and 28 KD) in the purified rat EPF fraction.

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.

Detection of Early Pregnancy Factor (EPF) in Mare Sera.

Early Pregnancy Factor (EPF) has been detected in the pregnant sera of almost all the species examined. Yet no EPF of mare sera had been reported prior to this study, because the method of examination had not yet been established. The present study was carried out to examine various factors which influence the rosette inhibition test of mares and to establish a reliable assay system for Rosette Inhibition Titre (RIT). The following items were found optimal for the detection of RIT in mare sera: human erythrocytes (red blood cells) showed the best reactivity among human, sheep, and rat erythrocytes; the ratio of lymphocytes to erythrocytes of 1:20 was proven to show better results; and Guinea Pig Complement (GPC) was not necessary. When the rosette inhibition test was performed on 10 pregnant and 9 nonpregnant sera, the RIT values ranged from 6 to 8 (7.2 ± 1.0) in nonpregnant sera, and from 9 to 12 (9.5 ± 1.0) in pregnant sera, with a significant difference (p<0.01). These results suggested the availability of the RIT for pregnant mare sera as an index of early detection of pregnancy in mare.

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

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

PAF released by preimplantation embryos binds to albumin.

The preimplantation embryo from a variety of mammalian species, including humans1, 2, mice3, 4, rabbit5 and sheep6, have been reported to release PAF in vitro. It has been demonstrated that embryo-derived PAF may act as an autocrine stimulant of embryonic metabolism and growth7, 8 and the release of PAF by human embryos created by in vitro fertilization appears to be correlated with embryonic viability and pregnancy potential1, 9, 10. PAF has a range of effects on maternal physiology which include: thrombocytopenia and splenic contraction11; modulation of prostaglandin F2a secretory activity of the uterus in vitro 12 and in vivo 13; changes in the vasculature of the uterine tube of mice14; and the induction of early pregnancy factor expression15.

Affinity Purification, Overexpression, and Characterization of Chaperonin 10 Homologues Synthesized with and without N-terminal Acetylation

Utilizing the ability of bacterial chaperonin 60 (GroEL) to functionally interact with chaperonin 10 (Cpn10) homologues in an ATP-dependent fashion, we have purified substantial amounts of mammalian, chloroplast, and thermophilic Cpn10 homologues from their natural host. In addition, large amounts of recombinant rat Cpn10 were produced in Escherichia coli and found to be identical to its authentic counterpart except for the lack of N-terminal acetylation. By comparing these two forms of Cpn10, it was found that acetylation does not influence the oligomeric structure of Cpn10 and is not essential for chaperone activity or mitochondrial import in vitro. In contrast, N-terminal acetylation proved crucial in the protection of Cpn10 against degradation by N-ethylmaleimide-sensitive proteases derived from organellar preparations of rat liver. The availability of large amounts of both affinity-purified and recombinant Cpn10 will facilitate not only further characterization of the eukaryotic folding machinery but also further scrutiny of the reported function of Cpn10 as early pregnancy factor.

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

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. 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. 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. 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.

Early pregnancy factor (EPF) as a marker for detecting subclinical embryonic loss in clomiphene citrate-treated women.

A discrepancy exists between the apparently normal ovulation and the pregnancy rates in women treated with clomiphene citrate (CC). Our previous studies have indicated that immuno-suppressive "early pregnancy factor" (EPF) is a novel marker to detect subclinical embryonic loss in infertile women. In the present study EPF was used as a marker to detect subclinical embryonic loss in women treated with CC with/without gonadotropins. In some of the women treated with CC, conception was assisted by artificial insemination with husband's semen (AIH). Our results have indicated that fertilization occurred (EPF + ve) in 47.7% (52/109) of women treated with CC with/without gonadotropins; 13.46% (7/52) retained the fetus and continued pregnancy till full term, whereas 78.9% (41/52) did not retain the fetuses. In the group where after stimulation, conception was assisted by AIH, fertilization was observed in 38.24% (26/68), retention in 11.54% (3/26) but subclinical embryonic loss was observed in 80.8% (21/26) cases. Thus, our results have indicated that subclinical embryonic loss may account for some of the discrepancy observed between the apparently normal ovulation and the pregnancy rates in women treated with clomiphene citrate.

Uterine and embryonic factors in early pregnancy

Uterine and embryonic factors in early pregnancy

EPF as a Marker for Early Embryonic Losses in Repeat Breeders.

Early pregnancy factor (EPF) is detectable in sera of pregnant cattle within 24 to 48 h after fertilization. It has been considered that the repeat breeder is relatively sterile as a result of fertilization failure and the early embryonic loss. This study used the rossete inhibition test to clarify whether repeat breeding is a result of early embyronic loss. We measured serum EPF in cattle that were diagnosed as repeat breeders to examine the incidence of early embryonic losses after artificial insemination (AI) (n=20). There were no significant differences in the presence of EPF on day 3 after AI for 20 repeat breeders and controls. Furthermore, EPF-positive rates decreased about 60% by day 21 after AI for both repeat breeders and a control group. It is suggested that a high incidence of early embryonic losses takes place in cattle, and we attempted embryo transfer (ET) as the treatment of choice.

Influence of Anti-Bovine Early Pregnancy Factor Antibody on Embryonic Development and Implantation in Rats.

To investigate whether early pregnancy factor (EPF) is necessary for embryonic development and implantation, anti-bovine EPF antibody was injected (2.5 mg/rat per injection) into pregnant rats intraperitoneally at 12, 36, 60 and 84 h after mating (total dose of the antibody; 10 mg). By 4.2 days post coitum (p.c.), 47.3% of embryos in the group of animals treated with anti-EPF antibody had developed to the blastocyst stage, compared with 97.2% and 95.4% in the control groups (injected with anti-nonpregnancy antibody and saline, respectively). Rats were administered with 2.5 mg of anti-EPF antibody on 0.5 and 1.5 day p.c.(postfertilization period: group 1), on 3.5 and 4.5 day p.c. (preimplantation period: group 2), and on 6.5 and 7.5 day p.c. (postimplantation period: group 3). When the animals were examined on 10 day p.c., the implantation rates in group 1 and group 2 had significantly decreased by 49.5% and 47.5%, respectively, compared with the control groups which were given injections of either anti-nonpregnancy antibody or saline. In group 3, however, the implantation rates had not been affected. Our data essentially agreed with the results of Igarashi [1] and Athanasas-Platsis et al. [2, 3] who investigated effects of anti-mouse or human EPF antibody in pregnant mice, and showed that anti-bovine EPF antibody crossreacted with rat EPF in vivo.

Early Pregnancy Factor: EPF-Like Substance(s) Purified from Pregnant Bovine Ovary and in vitro Fertilized Ovum Culture Medium

Early Pregnancy Factor: EPF-Like Substance(s) Purified from Pregnant Bovine Ovary and in vitro Fertilized Ovum Culture Medium

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 [H]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 (˜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. (Hepatology 1994;20:1294–1302).