Rosette Inhibition Test Research Articles

Recent Possibilities for the Diagnosis of Early Pregnancy and Embryonic Mortality in Dairy Cows.

Simple SummaryPregnancy diagnosis plays an essential role in decreasing days open in dairy farms; therefore, it is very important to select an accurate method for diagnosing early pregnancy. Besides traditional pregnancy diagnoses made by rectal palpation of the uterus from 40 to 60 days after AI and measuring the serum or milk progesterone concentration between 18 to 24 days after AI, there are several new possibilities to diagnose early pregnancy in dairy farms. However, it is very important to emphasize that before introducing any new diagnostic test, we need to make sure the accuracy of that particular test to be able to decrease the rate of iatrogenic pregnancy losses caused by prostaglandin or resynchronization treatments. This review focuses on the diagnostic possibilities and limitations of early pregnancy diagnosis in the field.One of the most recent techniques for the on-farm diagnosis of early pregnancy (EP) in cattle is B-mode ultrasonography. Under field conditions, acceptable results may be achieved with ultrasonography from Days 25 to 30 post-AI. The reliability of the test greatly depends on the frequency of the transducer used, the skill of the examiner, the criterion used for a positive pregnancy diagnosis (PD), and the position of the uterus in the pelvic inlet. Non-pregnant animals can be selected accurately by evaluating blood flow in the corpus luteum around Day 20 after AI, meaning we can substantially improve the reproductive efficiency of our herd. Pregnancy protein assays (PSPB, PAG-1, and PSP60 RIA, commercial ELISA or rapid visual ELISA tests) may provide an alternative method to ultrasonography for determining early pregnancy or late embryonic/early fetal mortality (LEM/EFM) in dairy cows. Although the early pregnancy factor is the earliest specific indicator of fertilization, at present, its detection is entirely dependent on the use of the rosette inhibition test; therefore, its use in the field needs further developments. Recently found biomarkers like interferon-tau stimulated genes or microRNAs may help us diagnose early pregnancy in dairy cows; however, these tests need further developments before their general use in the farms becomes possible.

Detection of early pregnancy in domestic ruminants

Tests for the detection of pregnancy early after insemination have not yet reached their full potential. Currently, the milk progesterone assay provides the earliest possible test, at an interval of one oestrous cycle after insemination, i.e. 17, 21 and 21 days in sheep, goats and cows respectively. This assay is pregnancy non-specific and rate of detection of pregnant animals is acceptable but less than desirable. Detection of activity of early pregnancy factor may develop into an excellent early test for many species, but the rosette inhibition test which is currently required has limited development and use. Pregnancy-specific protein B has been developed as a radioimmunoassay and is reliable under laboratory situations for ruminants. It can be used after 24 days of gestation in the cow. Application to field testing awaits development. Other pregnancy-associated or specific substances which are found in maternal body fluids might develop as pregnancy markers. Ultrasonic devices might provide very early detection in cattle but the expense of a test will limit application. All tests for pregnancy early after insemination have an inherent inaccuracy. Presence of an embryo at the time the test is applied will not assure pregnancy at the time of a confirmatory test, such as birth of live young or rectal examination in cows after 35 days of gestation. Therefore, no matter how early the test, a follow-up examination might be desirable in intensively managed herds or flocks. The animal industry is on the verge of new biotechnological approaches to reproductive management. The potential seems as great as the imagination.

Identification of heat shock protein 10 within the equine embryo, endometrium, and maternal peripheral blood mononuclear cells

Early pregnancy factor has been identified as a 10-kDa extracellular homolog of heat shock protein 10 (Hsp10). Hsp10 has been detected during early pregnancy in serum of mice, sheep, pigs, horses, cows, and humans by the rosette inhibition test. Hsp10 has also been associated with several neoplastic and autoimmune diseases. The goal of the present study was to determine if Hsp10 could be detected in the early equine embryo through the use of immunohistochemistry and quantitative real-time PCR. Additionally, analysis of systemically harvested peripheral blood mononuclear cells (PBMCs) from both pregnant and nonpregnant mares was evaluated to determine expression levels of HSP10. Embryos were collected from Quarter Horse mares by uterine lavage at either 8 or 25 days after ovulation. Collection and separation of PBMCs occurred on Day 8 for both pregnant and nonpregnant mares. Immunohistochemistry revealed cytoplasmic localization of HSP10 throughout the single layer of ectodermal cells forming the trophoblast in Day-8 embryos. Day-25 embryos demonstrated intense localization focally along the apical border of ectodermal cells forming the trophoblast layer of the developing chorion. There was no nuclear staining in either embryonic population. Quantitative real-time PCR detected the presence of mRNA for HSP10 in both 8- and 25-day equine embryos. Day-25 embryos exhibited an elevated degree of expression (P = 0.006) compared with the 8-day embryos for HSP10. Endometrial samples did not display any significant difference in degree of expression for HSP10 (P = 0.10). Finally, PBMCs from pregnant mares demonstrated elevated (P = 0.03) expression of HSP10 compared to the nonpregnant mares on Day 8 of the estrous cycle. This study confirmed the presence of HSP10 protein and mRNA expression of HSP10 in equine embryos at two maturation stages. Additionally, the presence of increased gene expression within PBMCs of pregnant mares suggests communication, possibly leading to necessary immunomodulatory effects between the embryo and mare.

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.

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.

Partial purification of mare early pregnancy factor.

Early pregnancy factor (EPF) is an immunosuppressive protein detected in the serum in early pregnancy. We have already reported the development of the rosette inhibition test for mare EPF and have detected EPF in thoroughbreds and ponies. Here, we attempted to purify equine EPF from pregnant mare serum. Mare EPF was purified by ultrafiltration and ion-exchange chromatography. Purified EPF was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting and a neutralization test. EPF activity was estimated as the rosette inhibition titer (RIT) by the rosette inhibition test. Purified EPF bound to carboxymethyl (CM) sepharose and did not adsorb to diethylaminoethyl (DEAE) sepharose. SDS-PAGE revealed that in the final purified fraction there were many proteins. In the immunoblotting analysis, a protein band of 25.8 kDa was detected as the pregnancy-specific band. Further, antibody gained from the 20 to 30 kDa protein band of the final purified fraction neutralized the RIT activity of pregnant mare serum. Mare EPF was detected in the final purified fraction and had a molecular weight of 25.8 kDa. EPF in the mare is similar to that obtained from the serum of pregnant cows.

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 ∼42 and 36 mg EPF from 300 ml bacterial and 1 L Sf9 cultures, respectively. The preparations were highly purified (⩾99% purity on SDS–PAGE for the bacterial products and ⩾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.

A protective effect of early pregnancy factor on experimental autoimmune encephalomyelitis induced in Lewis rats by inoculation with myelin basic protein

Experimental autoimmune encephalomyelitis (EAE) is an organ-specific autoimmune disease characterised by inflammation and demyelination of the central nervous system and is the best available animal model of multiple sclerosis (MS). Since previous studies have shown that EAE is less severe or is delayed in onset during pregnancy and that administration of the pregnancy hormone early pregnancy factor (EPF) down-regulates EAE, experiments in the present study were designed to explore further the role of EPF in EAE. By using the rosette inhibition test, the standard bioassay for EPF and, by semi-quantitative RT-PCR techniques, we have now shown that inflammatory cells from the spinal cord of rats with EAE can produce and secrete EPF, with production being greatest during recovery from disease. Administration of EPF to rats with EAE resulted in a significant increase in the expression of IL-4 and IL-10 mRNA and a significant decrease in IFN-γ mRNA expression in spinal cord inflammatory cells. Encephalitogenic MBP-specific T cell lines were prepared from popliteal lymph nodes of rats with EAE. Proliferation assays using these cells demonstrated the ability of exogenous EPF to down-regulate the responses of T lymphocytes to MBP.

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.

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.

Study of early pregnancy factor (EPF) in equine (Equus caballus).

Early pregnancy factor (EPF) is an immunosuppressive protein detected in the early pregnancy serum. We have already reported that we developed the rosette inhibition test for mare EPF and detected EPF in thoroughbreds. The aim of this study was to determine whether or not our method could be used clinically. The rosette inhibition test for equine EPF was carried out on serum from six nonpregnant and six pregnant Shetland ponies, a female and a male Chinese pony, and four nonpregnant and 13 pregnant thoroughbred mares. In the thoroughbreds sera were collected during the pregnancy period. Furthermore, we measured progesterone and detected pregnant mare serum gonadotrophin (PMSG) in order to confirm pregnancy of the Chinese pony 3 and 6 months after mating. In the nonpregnant Shetland ponies, the rosette inhibition titre (RIT) was 6.0+/-1.0 and EPF was negative. In contrast, in the pregnant ponies, the RIT was 9.2+/-0.4 and EPF was positive. Based on these results, we diagnosed pregnancy of the Chinese pony. The RIT of the female Chinese pony (3 months after mating) was above 10 and EPF was positive. Furthermore, we detected PMSG and progesterone in the serum of this pony. EPF appeared in the maternal blood circulation at 24-72 hr after mating, it was detected until the second trimester, and after that it disappeared from the maternal serum. The pony's EPF was detected by using the same rosette inhibition test as in the thoroughbred and was present from 24 to 72 hr after mating until the second trimester. The results indicated that our method was useful for pregnancy diagnosis of Equine.

Early pregnancy factor: an extracellular chaperonin 10 homologue.

Early pregnancy factor (EPF) has been identified as a homologue of chaperonin 10 (cpn10) with immunosuppressive and growth factor properties. As a homologue of cpn10, it belongs to the heat shock family of proteins (hsp) but, unlike other members of this family, EPF is detected extracellularly. Early pregnancy factor was first discovered in pregnancy serum by the rosette inhibition test, and the novelty of its discovery was that its presence could diagnose pregnancy within 6-24 h of a fertile mating. As well as being a monitor of the presence of a viable embryo, it is necessary for embryonic survival. In this capacity it acts as both an immunosuppressant and growth factor. Early pregnancy factor is also a product of proliferating primary and neoplastic cells and functions as an autocrine growth factor both in vivo and in vitro. It has a modifying effect on the outcome of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Early pregnancy factor is considered to be one of the major factors involved in the modification of multiple sclerosis observed during 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.

Detection of Early Pregnancy Factor in the Sera of Cows Whose Uteri were Infused with Trophoblastic Cell Culture Medium.

The aim of this study was to examine whether or not uterine infusion with a medium showing positive early pregnancy factor (EPF) activity influences maternal physiology in cattle. Trophoblastic cells derived from 12-day-old bovine embryos were cultured in Dulbecco's PBS (+) at 39 C in air for 24 h. EPF activity was assayed using the rosette inhibition test. A rosette inhibition titer (RIT) above 4 was regarded as indicating positive EPF activity. The trophoblastic cell culture medium (Tro-CM) showed positive EPF activity. On days 7 to 12 after estrus, five cows each received a nonsurgical infusion of Tro-CM (0.25 ml) in the lumen of the uterine horn. Blood samples were collected daily by the tail artery or vein puncture from day 0 (the day of infusion) to day 5. All five cows showed positive EPF activity in their sera on days 2 to 4, and three cows out of the five kept showing positive EPF activity for 2 to 3 days. The result demonstrated that sera of cows infused with a small amount of EPF-positive medium (Tro-CM) into the uterus showed temporary positive EPF activity in maternal serum, further suggesting that EPF released from Tro-CM would induce a release of maternal EPF, but not by directly entering into the maternal blood flow.

Induction of early pregnancy factor activity in vitro by platelet-activating factor in mice.

The rosette inhibition test was used to determine early pregnancy factor activity in culture media from oestrous mouse ovaries and oviducts stimulated in vitro for early pregnancy factor production under different experimental conditions. Embryo conditioned media, platelet-activating factor and cortical granule release media could all stimulate the production of early pregnancy factor by oestrous mouse ovaries and oviducts. This stimulation was completely blocked by the presence of BN 52021, a platelet-activating factor receptor antagonist. This study indicates that platelet-activating factor is the 'ovum factor' released by the zygote on fertilization to initiate the synthesis of early pregnancy factor.

Bovine Early Pregnancy Factor in Recipient Serum with Embryo Transfer on the Day More Than 5 Days Different from the Estrus Cycle of Donor

Bovine early pregnancy factor (EPF) has been detected in the recipient serum after embryo transfer which was performed on day 7 to 8 after estrus. However, there have been no reports of the detection of EPF in the recipient serum with embryo transfer on the day more than 3 days different from the estrous cycle of the donor. The aim of this study was to determine if EPF is detectable from serum of Holstein cow recipients with embryo transfer on day 12 to 16 after estrus which was 5 to 9 days earlier than that of the donor. Embryos were collected nonsurgically from superovulated Holstein cows on day 7 to 8 after artificial insemination (AI). The embryos were transferred nonsurgically to seven recipients on day 12 to 16 after estrus. Blood samples of the recipients were collected on day 0 (the day of embryo transfer) to 15. EPF activity was assayed by the rosette inhibition test. A rosette inhibition titer (RIT) above 4 was regarded as indicating positive EPF activity. EPF activity was positive in the serum of seven recipients on day 2 to 3 after embryo transfer. Subsequently, six recipients out of seven remained positive for 2 to 3 days. As for the other recipient, positive EPF activity was maintained until day 15 and then a female calf was delivered on day 268 after embryo transfer. These results provided evidence that EPF was detected in the serum of recipients of transferred embryos and was indicative of embryonic life (or existence of the embryo) on even day 12 to 16 after estrus, which was 5 to 9 days earlier than that of the donor

Synthesis of early pregnancy factor using red deer (Cervus elaphus) as a delayed implantation model.

This study measured serum early pregnancy factor (EPF) in pregnant red deer (Cervus elaphus) and ascertained whether EPF synthesis is associated with implantation. Serial serum samples were taken from mated hinds up to 42 days postconception and analyzed for EPF activity using the rosette inhibition test. EPF activity was then correlated with calving records and stages of preimplantation development. EPF was detected in all pregnant animals, with a twin pregnancy giving increased EPF activity. Three animals gave an EPF response following fertilization but failed to continue beyond the preimplantation embryo stage. The increase in EPF synthesis previously associated with implantation in other mammals occurred at the blastocyst stage in red deer. EPF synthesis in red deer (Cervus elaphus) is consistent with the preimplantation period, as occurs in other mammals. However, the second phase of the biphasic increase in early pregnancy factor production is associated with blastocyst formation, not implantation.

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 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.