Progress 07/15/09 to 07/14/12
Outputs
OUTPUTS: Focal adhesion kinase (FAK), also known as protein tyrosine kinase 2, is localized to focal adhesions and is a key component of signal transduction pathways mediated by integrins. The presence of FAK at the mRNA and protein levels in bovine oocytes was verified by real time PCR (qPCR) and immunoprecipitation and the localization of FAK at the site of sperm binding to the oocyte plasma membrane was verified using immunohistochemistry. Small interfering RNA (siRNA) duplexes directed against bovine FAK, and known FAK inhibitors FAK I and FAK II, were microinjected into bovine oocytes at various concentrations and the resulting effects on FAK mRNA and protein levels, intracellular calcium release, and embryo development were evaluated. FAK I inhibitor, FAK II inhibitor, all resulted in lower cleavage rates and a decrease in the number of oocytes responding with calcium transients. The sham and scrambled FAK sequence siRNA control microinjections had no effect on fertilization while the microinjection of the FAK siRNA did result in significantly lower cleavage rates and the FAK siRNA treatment group was not significantly different than the no sperm control. The microinjection of FAK siRNA resulted in lower levels of FAK mRNA transcript while western blot analysis indicated a decrease in the relative levels of FAK protein corresponding to a decrease in mRNA levels after microinjection with the FAK siRNA. These data are compelling evidence that FAK is involved in bovine activation and fertilization. We have also conducted research that has lead to discovery of how early clone cattle pregnancy are lost. We have published three refereed journal articles and given eight presentations at national and international scientific meetings. PARTICIPANTS: Ken White - Utah State University, ADVS, CIB Chris Davies, Utah State University, ADVS, CIB Kamal Rashid, Utah State University, ADVS, CIB Lee Rickords, Utah State University, ADVS, CIB Jacob Parnell, Utah State University, Biology, CIB TARGET AUDIENCES: Reproductive Biology Biotechnology Reproductive Immunology Animal Genomics Environmental Genomics PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Currently there is a lack of information regarding how the sperm initiates development in cattle after fertilization. This project has result in a much better understanding of how the egg, once fertilized by the sperm cell, begins to grow. Cloned embryos are produced without fertilization occurring and the egg must be "triggered" to start growing by artificial means. The information obtained through this project is critical to helping to increase the number of cloned cattle embryos that will produce live offspring.
Publications
- No publications reported this period
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Progress 07/15/10 to 07/14/11
Outputs
OUTPUTS: Intracellular calcium (Ca2+i) release, a hallmark of oocyte activation, is the end result of a complex series of signal transduction pathways, which have yet to be completely characterized in the bovine model. It is well known that src family kinases (SFK) activate Phospholipase C (PLC) which converts phosphatidyl inositol (4,5)-bisphosphate into diacylglycerol and 1,4,5-inositol trisphosphate (IP3) which is directly involved in releasing Ca2+i from the endoplasmic reticulum. A two-prong approach was undertaken to identify the mechanisms involved in the signal transduction pathway resulting in bovine activation. The first approach utilized immunoblotting to identify the presence of endogenous PLC isoforms in total bovine oocyte lysate using primary antibodies against four distinct isoforms of PLC. Immunoblotting was performed according to standard laboratory protocols and confirmed the presence of several PLC isoforms. The second approach involved the microinjection of the four corresponding PLC primary antibodies into in vitro matured bovine oocytes at a 1:100 dilution in a 8pL volume. A control injection of ddH20 was used to assist in evaluation of oocyte quality following inject and to serve as a control. Following microinjection, oocytes were fertilized and cultured in vitro according to standard laboratory procedures (Reed et al., 1996). Activation and development were assessed by recording cleavage at 48 hours post fertilization. Results from these experiments were presented at the American Cell Biology Association meetings in December, 2009. Experimental results were also presented at the 2010 annual meetings of the International Embryo Transfer Society. Results were also presented in Beijing China in the Join US-China Animal and Dairy Science meetings and in workshops in Hohhot and Tian China in November 2009. PARTICIPANTS: Ken White - Utah State University, ADVS, CIB; Chris Davies, Utah State University, ADVS, CIB; Kamal Rashid, Utah State University, ADVS, CIB; Lee Rickords, Utah State University, ADVS, CIB; Jacob Parnell, Utah State University, Biology, CIB; Giovanni Rompato, Utah State University, CIB TARGET AUDIENCES: Reproductive Biology; Biotechnology; Reproductive Immunology; Animal Genomics PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Microinjection of several PLC-specific antibodies resulted in no effect on cleavage rates while others essentially completely blocked development. Determining the presence and involvement of these intracellular signaling molecules in bovine oocyte fertilization and activation will result in better activation protocols for nuclear transfer (NT) embryos and ultimately improve the efficiency of the NT process along with facilitating study of specific aspects of activation by identifying messages associated with the process. Deficiencies in the success rate of somatic cell nuclear transfer are widely held to be epigenetic in nature, and arise from the limited ability of a differentiated donor cell to erase epigenetic signatures required for nuclear reprogramming. Following bovine somatic cell nuclear transfer DNA methylation signatures of two genes necessary for pluripotency and self-renewal, namely Nanog and POU5F1 (Oct-4) more closely resemble that of somatic cells rather than In Vitro fertilized embryos. A retained methylation signature following scNT likely leads to interaction with methyl binding domain proteins capable of binding to methylated promoter regions and acting to silence gene expression. Using a modified version of Electrophoretic Mobility Shift Assay (EMSA) targeted to the genes Nanog and POU4F1 (Oct-4) we identified DNA binding proteins that interact specifically with the methylated promoter regions. We observed protein binding specific to a methylated DNA template that differs from the binding proteins specific to the non-methylated DNA template. These findings indicate that the gene promoter region is acted upon by different DNA binding proteins depending on its methylation status, and that the retention of a hypermethylation signature could lead to the premature down-regulation of the genes Nanog and POU5F1.
Publications
- Stevens, J. R., Bell, J. L., Aston, K. I., & White, K. L. 2010. A Comparison of Probe-Level and Probeset Models for Small-Sample Gene Expression Data: BMC Bioinformatics, 11(281).
- Aston, K. I., Li, G. P., Hicks, B. A., Sessions, B. R., Bunch, T. D., Rickords, L. F., Weimer, B., & White, K. L. 2010. Aberrant Expression of Developmentally Important Genes in Various Stages of Preimplantation Bovine Somatic Cell Nuclear Transfer Embryos. Cloning and Stem Cells.: Cloning and Stem Cells, 12: 23-32.
- White, K. L., Pate, B. J., & Sessions, B. R. 2010. Oolemma Receptors and Oocyte Activation: Sys. Biol. Reprod. Med., 56: 365-375.
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Progress 07/15/09 to 07/14/10
Outputs
OUTPUTS: Intracellular calcium (Ca2+i) release, a hallmark of oocyte activation, is the end result of a complex series of signal transduction pathways, which have yet to be completely characterized in the bovine model. It is well known that src family kinases (SFK) activate Phospholipase C (PLC) which converts phosphatidyl inositol (4,5)-bisphosphate into diacylglycerol and 1,4,5-inositol trisphosphate (IP3) which is directly involved in releasing Ca2+i from the endoplasmic reticulum. A two-prong approach was undertaken to identify the mechanisms involved in the signal transduction pathway resulting in bovine activation. The first approach utilized immunoblotting to identify the presence of endogenous PLC isoforms in total bovine oocyte lysate using primary antibodies against four distinct isoforms of PLC. Immunoblotting was performed according to standard laboratory protocols and confirmed the presence of several PLC isoforms. The second approach involved the microinjection of the four corresponding PLC primary antibodies into in vitro matured bovine oocytes at a 1:100 dilution in a 8pL volume. A control injection of ddH20 was used to assist in evaluation of oocyte quality following inject and to serve as a control. Following microinjection, oocytes were fertilized and cultured in vitro according to standard laboratory procedures (Reed et al., 1996). Activation and development were assessed by recording cleavage at 48 hours post fertilization. Results from these experiments were presented at the American Cell Biology Association meetings in December, 2009. Experimental results were also presented at the 2010 annual meetings of the International Embryo Transfer Society. Results were also presented in Beijing China in the Join US-China Animal and Dairy Science meetings and in workshops in Hohhot and Tian China in November 2009. PARTICIPANTS: Ken White - Utah State University, ADVS, CIB Chris Davies, Utah State University, ADVS, CIB Kamal Rashid, Utah State University, ADVS, CIB Lee Rickords, Utah State University, ADVS, CIB Jacob Parnell, Utah State University, Biology, CIB Giovanni Rompato, Utah State University, CIB TARGET AUDIENCES: Reproductive Biology Biotechnology Reproductive Immunology Animal Genomics Environmental Genomics PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Microinjection of several PLC-specific antibodies resulted in no effect on cleavage rates while others essentially completely blocked development. Determining the presence and involvement of these intracellular signaling molecules in bovine oocyte fertilization and activation will result in better activation protocols for nuclear transfer (NT) embryos and ultimately improve the efficiency of the NT process along with facilitating study of specific aspects of activation by identifying messages associated with the process. Deficiencies in the success rate of somatic cell nuclear transfer are widely held to be epigenetic in nature, and arise from the limited ability of a differentiated donor cell to erase epigenetic signatures required for nuclear reprogramming. Following bovine somatic cell nuclear transfer DNA methylation signatures of two genes necessary for pluripotency and self-renewal, namely Nanog and POU5F1 (Oct-4) more closely resemble that of somatic cells rather than In Vitro fertilized embryos. A retained methylation signature following scNT likely leads to interaction with methyl binding domain proteins capable of binding to methylated promoter regions and acting to silence gene expression. Using a modified version of Electrophoretic Mobility Shift Assay (EMSA) targeted to the genes Nanog and POU4F1 (Oct-4) we identified DNA binding proteins that interact specifically with the methylated promoter regions. We observed protein binding specific to a methylated DNA template that differs from the binding proteins specific to the non-methylated DNA template. These findings indicate that the gene promoter region is acted upon by different DNA binding proteins depending on its methylation status, and that the retention of a hypermethylation signature could lead to the premature down-regulation of the genes Nanog and POU5F1.
Publications
- Latta, L., Baker, M., Crowl, T. Parnell, Jacob, Weimer, B.; DeWald, D., Pfrender, M. 2010. Species and Genotype Diversity Drive Community and Ecosystem Properties in Experimental Microcosms. Evolutionary Ecology, USA.
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