Progress 09/01/09 to 08/31/12
Outputs
OUTPUTS: The most important output from this work was an understanding of the mechanism by which CSF2 (previously called GM-CSF) can act on the preimplantation embryo to affect developmental events much later in gestation. Exposure of embryos to CSF2 from Day 5-7 of development improves the ability of embryos to establish pregnancy after transfer to recipients and, moverover, reduces the probability that established pregnacies are lost later in development. Our work implicates changes in gene expression, particularly the WNT system and genes involved in apoptosis, in these effects. An additional outcome was the development of a procedure to separate the two major cell types ofthe blastocyst-stage embryo (inner cell mass cells and trophectoderm cells) based on magnetic-activated cell sorting. This technique could have wide use in embryology research. Results from the work were disseminated to the research community through 5 research papers and two review articles. In addition, implications of the research for embryo transfer was disseminated through the presentation of invited lectures at various research institutions (University of Missouri, Utah State University, USDA-ARS, Beltsville, University of Kentucky, and University of Bonn) companies (Pfizer Animal Health and Minitube), producer meetings in Arizona, Peru, and the UK, and at meetings of the International Embryo Transfer Society, Society for Reproduction and Fertility, European Society for Domestic Animal Reproduction, Southern Ontario reproductive Biology Group, European Society for Reproductive Immunology and the Banff Symposium on Functional Genomics of Early Development in Livestock. PARTICIPANTS: PJ Hansen Barbara Loureiro (received PhD) Aline Bonilla (received PhD) Manabu Ozawa (postdoctoral training) Kyle Dobbs (studying for PhD) Mateus Sudano (studying for PhD) TARGET AUDIENCES: Target audiences included fellow scientists (reached through publications and invited presentations), people working in the embryo technologies field (reached through invited presentations at companies and through informal discussion) and people in the livestock industry (reached through extension meetings and informal discussion). PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The work is important because CSF2, or the other growth factor studied, insulin-like growth factor-1, is likely to prove useful in improving the pregnancy rate achieved after embryo transfer in both cattle and women. Currently, the opportunities available through use of embryo transfer (improved rate of genetic progress and enhanced fertility) are limited by suboptimal embryonic survival. Not only might we improve outcomes of emrbyo transfer by incorporating CSF2 or IGF1 in culture media, the understanding of the mechanisms by which CSF2 precent embryonic loss will lead to new approaches for enhancing fertility in cattle in general. For example, we are currently evaluating single nucleotide polymorphisms in genes regulated by CSF2 in the embryo to identify possible genetic markers of fertility that could be selected for to enhance genetic merit for fertility.
Publications
- Ozawa, M., Sakatani, M., Yao-J-Q, Shanker, S., Yu, F., Yamashita, R., Wakabayashi, S., Nakai, K., Dobbs, K.B., Sudano, M.J., Farmerie, W.G., and Hansen, P.J. 2013. Global gene expression of the inner cell and trophectoderm of the bovine blastocyst. submitted.
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: One experiment was performed to understand the mechanism by which CSF2 increases embryonic and fetal survival. Conceptuses were produced in vitro in the presence or absence of 10 ng/ml CSF2 from Day 5 to 7 after insemination, transferred into cows, and flushed from the uterus at Day 15 of pregnancy. There was a tendency (P=0.07) for the proportion of cows with a recovered conceptus to be greater for those receiving a CSF2 treated conceptus (35% for control vs. 66% for CSF2). Antiviral activity in uterine flushings, a measure of the amount of interferon-tau (IFNT2) secreted by the conceptus, tended to be greater for cows receiving CSF2-treated conceptuses than for cows receiving control conceptuses. This difference approached significance when only cows with detectable antiviral activity were considered (P=0.07). In addition, CSF2 increased mRNA for IFNT2 (P=0.08) and keratin 18 (P<0.05) in extraembryonic membranes. Among a subset of filamentous conceptuses that were analyzed by microarray hybridization, there was no effect of CSF2 on gene expression in the embryonic disc or extraembryonic membranes. In other experiments, we evaluated the function of another maternal growth factor, insulin-like growth factor-1. We demonstrated that IGF1 increased development to the blastocyst stage by acting through a MAPK pathway from Day 4-8 of development. Moreover, IGF1 could protect embryos from heat shock if heat shock occurred at Day 5 of development (after genome activation) but not it occurring at the two-cell stage of development. Finally, a new method to separate inner cell mass cells from trophectoderm cells was developed. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: The only modification was an extension of our focus on maternally derived growth factors to also examine IGF1 effects on the embryo.
Impacts
Previous results indicates that CSF2 treatment during the preimplantatin period can promote survival of the embryo after transfer into recipients. The outcomes from the recent experiments suggest that the increase in calving rate caused by CSF2 treatment involves, in part, more extensive development of extraembryonic membranes and capacity of the conceptus to secrete IFNT2 at Day 15 of pregnancy. Experiments with IGF1 could lead to incorporation of IGF1 into culture media to increase the yield of embryos from in vitro production systems and to increase embryonic resistance to heat stress. The new technique for separating inner cell mass cells from trophectoderm is a useful tool for understanding embryonic development.
Publications
- Loureiro, B., Oliveira, L.J., Favoreto, M.G., and Hansen, P.J. (2011) Colony-stimulating factor 2 inhibits induction of apoptosis in the bovine preimplantation embryo. Am. J. Reprod. Immunol. 65, 578-588.
- Bonilla, A.Q.S., Oliveira, L.J., Ozawa, M., Newsom, E.M., Lucy, M.C., and Hansen, P.J. (2011) Developmental changes in thermoprotective actions of insulin-like growth factor-1 on the preimplantation bovine embryo. Mol. Cell. Endocrinol. 332, 170-179. Ozawa, M., and Hansen, P.J. (2011) A novel method for purification of inner cell mass and trophectoderm cells from blastocysts using magnetic activated cell sorting. Fertil. Steril. 95, 799-802.
- Loureiro, B., Block, J., Favoreto, M.G., Carambula, C., Pennington, K.,A., Ealy, A.D., and Hansen, P.J. (2011) Consequences of conceptus exposure to colony stimulating factor 2 on survival, elongation, interferon-t secretion and gene expression. Reproduction 141, 617-624. Bonilla, A.Q.S., Ozawa, M., and Hansen, P.J. (2011) Timing and dependence upon mitogen-activated protein kinase signaling for pro-developmental actions of insulin-like growth factor 1 on the preimplantation bovine embryo. Growth Hormone IGF Res. 21, 107-111.
- Hansen, P.J. (2011) The immunology of early pregnancy in farm animals. Reprod. Dom. Anim. 46 (Suppl. 3), 18-30.
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: Addition of CSF2 to culture medium increases post-transfer survival of bovine embryos. Here we provide evidence that one mechanism by which CSF2 affects the embryo is through inhibition of apoptosis. In the first experiment, genes and pathways whose expression were regulated by CSF2 were identified by microarray analysis. Embryos were treated with 10 ng/ml CSF2 or vehicle at Day 5 after insemination; morulae were selected for microarray analysis at Day 6. A total of 214 genes were differentially regulated and 160 of these could be annotated (67 upregulated genes and 93 downregulated genes). Differentially expressed genes could be placed in 13 biological process ontologies in four functional groups (development and differentiation process, cell communication, apoptosis and cell adhesion). Antiapoptotic effects of CSF2 were confirmed in a second experiment. Embryos were treated with CSF2 or vehicle at Day 5. On day 6 (24 h after treatment), morulae were cultured for 15 h at either 42 C (a temperature that induces apoptosis) or 38.5 C (cow body temperature). The magnitude of the increase in TUNEL positive cells caused by heat shock was reduced by CSF2. In conclusion, CSF2 blocks apoptosis in bovine embryos through actions associated with regulation of genes controlling apoptosis. PARTICIPANTS: PJ Hansen and James Resnick(PI), Jeremy Block (cooperating scientist), Luciano Bonilla (technician), Barbara Loureiro and Aline Bonilla (graduate students). Most of Loureiro's PhD dissertation represents work funded by the grant. TARGET AUDIENCES: The main audience currently is the international scientific community, which is being reached through publications, invited talks and presentations at scientific meetings. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
These results suggest that apoptosis can be an important determinant of embryonic survival. In addition, data on the gene expression patterns suggest that the process of gastrulation is regulated by maternal signals and that failure of proper regulation may lead to pregnancy failure.
Publications
- No publications reported this period
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