Progress 09/01/11 to 08/31/16
Outputs
Target Audience:The primary target audience are research scientists with a focus on embryology, reproductive biology, pregnancy biology, stem cell biology, and reproductive immunology. The results of the research are also relevant to dairy farmers, cattle ranchers, and embryo technology practitioners. Efforts will be made at the appropriate time to convey results and recommendations for changes in reproductive management and embryo production to these groups. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In the project period, research contributed to the doctoral research programs of three students, the masters research program of one student, and to the research program of an intern from Brazil. How have the results been disseminated to communities of interest?Seminar: Exploitation of developmental programming in the preimplantation period to enhance fertility and improve postnatal function in cattle. Zoetis, Kalamazoo, MI, November 4, 2015. What do you plan to do during the next reporting period to accomplish the goals?The project is complete but wewill finish an experiment to evaluate whether CSF2 treatment from day 5-7 after insemination alters the characteristics of thefetus and placenta at Day 90 of gestation.
Impacts
What was accomplished under these goals?
Colony stimulating factor 2 can have multiple effects on the function of the preimplantation embryo that include increased potential to develop to the blastocyst stage, reduced apoptosis, and enhanced ability of inner cell mass (ICM) to remain pluripotent after culture. Recently, we identified genes regulated by CSF2 in the ICM and trophectoderm (TE) of the bovine blastocyst with the goal of identifying possible molecular pathways by which CSF2 increases developmental competence for survival. Embryos were produced in vitro and cultured from Day 6 to 8 in serum-free medium containing 10 ng/ml recombinant bovine CSF2 or vehicle. Blastocysts were harvested at Day 8 and ICM separated from TE by magnetic-activated cell sorting. RNA was purified and used to prepare amplified cDNA, which was then subjected to high-throughput sequencing using the SOLiD 4.0 system. Three pools of amplified cDNA were analyzed per treatment. The number of genes whose expression was regulated by CSF2, using P < 0.05 and >1.5-fold difference as cut-offs, was 945 in the ICM (242 upregulated by CSF2 and 703 downregulated) and 886 in the TE (401 upregulated by CSF2 and 485 downregulated). Only 49 genes were regulated in a similar manner by CSF2 in both cell types. The three significant annotation clusters in which genes regulated by ICM were overrepresented were related to membrane signaling. Genes downregulated by CSF2 in ICM were overrepresented in several pathways including those for ERK and AKT signaling. The only significant annotation cluster containing an overrepresentation of genes regulated by CSF2 in TE was for secreted or extracellular proteins. In addition, genes downregulated in TE were overrepresented in TGFβ and Nanog pathways. Conclusions: Differentiation of the blastocyst is such that, by Day 8 after fertilization, the ICM and TE respond differently to CSF2. Analysis of the genes regulated by CSF2 in ICM and TE are suggestive that CSF2 reinforces developmental fate and function of both cell lineages. In vitro production (IVP) of embryos can disrupt fetal and placental development and increase risk of abnormal fetal growth. Maternal factors play a role in developmental programming of the early embryo. Colony-stimulating factor 2 (CSF2) is present in the oviduct and endometrium and has improved competence of the preimplantation embryo to establish pregnancy in cattle. Another experiment was performed with the objective of determining whether CSF2 during embryo culture alters fetal development and alleviates abnormalities associated with IVP. Holstein oocytes were matured and fertilized in vitro with Xsorted semen from a Holstein bull. Putative zygotes were cultured in SOF-BE1 at 5% CO2 and 5% O2 for 5 days and then randomly assigned to receive vehicle (IVP-control) or 10 ng/mL CSF2 (IVP-CSF2). Grade I blastocysts were transferred on day 7 to Holstein recipients that were previously randomized to receive an IVP-control or an IVP-CSF2 embryo. A third group of cows included in the randomization was assigned to be artificially inseminated on day 0 using the same bull as for IVP (AI). Pregnancy was terminated on day 85 or 86. Statistical analysis was performed by analysis of variance using the GLM procedure of SAS with contrasts for AI vs (IVP-control+IVP-CSF2) (contrast 1; C1) and IVP-control vs IVP-CSF2 (contrast 2; C2). A total of 23 morphometric measurements of placenta and fetus were made on 9 AI, 12 IVP and 7 CSF2 female singletons. Conceptuses derived by IVP (IVP-control and IVP-CSF2) differed from those derived by AI for four characteristics including fetal body weight (142.9 ± 4.7, 157.2 ± 4.4, and 162.6 ± 6.1 g for AI, IVP-control and IVP-CSF2, respectively; C1, P=0.0237), eviscerated weight (102.9 ± 3.4, 113.6 ± 3.2, and 112.2 ± 4.4 g; C1, P=0.0602), crown-rump length (CRL) (13.7 ± 0.2, 14.0 ± 0.2, and 14.7 ± 0.3 g; C1, P=0.0434; C2, P=0.0631) and umbilical cord diameter (0.85 ± 0.08, 1.1 ± 0.08, and 0.91 ± 0.1 cm; P=0.0519). Note that while IVP-CSF2 conceptuses were generally similar to those for IVP-control, CRL tended to be highest for IVP-CSF2. Also, umbilical cord diameter for IVP-CSF2 was similar to AI and lower than IVP-control. Data from one fetus in the IVP-CSF2 group was excluded from analysis because it had a phenotype consistent with large offspring syndrome. Body weight (354 g) was two-fold larger than other fetuses (average = 155 g) and placental weight was seven-fold greater (1,505 vs 211 g). In addition, organs were enlarged and severe ascites and hemorrhagic cotyledons were observed. In conclusion, IVP resulted in increased fetal size and umbilical cord diameter without other significant effects on placental morphometry. CSF2 did not alleviate adverse effects of culture on fetal growth, exacerbating effects on CRL, but did reduce effects of IVP on umbilical cord diameter. Gene expression analysis in fetal liver, muscle, and placenta is underway to further characterize effects of CSF2.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Denicol, A.C., Le�o, B.C.S., Dobbs, K.B., Mingoti, G.Z., and Hansen, P.J. (2015) Influence of sex on basal and dickkopf-1 regulated gene expression in the bovine morula. Plos One 10, e0133587.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2015
Citation:
Kannampuzha-Francis, J., Denicol, A.C., Loureiro, B., Kaniyamattam, K., Ortega, M.S., and Hansen, P.J. (2015) Exposure to colony stimulating factor 2 during preimplantation development increases postnatal growth in cattle. Mol. Reprod. Dev. 82, 892-897.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2016
Citation:
Ozawa, M., Sakatani, M., Dobbs, K.B., Kannampuzha-Francis, J., and Hansen, P.J. (2016) Regulation of gene expression in the bovine blastocyst by colony stimulating factor 2. BMC Res. Notes 9, 250.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Chen, Z., Hagen, D.E., Wang, J., Elsik, C.G., Tieming, J., Siqueira, L.G., Hansen, P.J., and Rivera, R.M. (2016) Global assessment of imprinted gene expression in the bovine conceptus by next generation sequencing. Epigenetics 11, 501-516.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2016
Citation:
Kannamphuza-Francis, J., Tribulo, P., and Hansen, P.J. (2016) Actions of activin A, connective tissue growth factor, hepatocyte growth factor and teratocarcinoma-derived growth factor 1 on development of the bovine embryo. Reprod. Fertil. Dev., in press.
|
Progress 09/01/15 to 08/31/16
Outputs
Target Audience:The primary target audience are research scientists with a focus on embryology, reproductive biology, pregnancy biology, stem cell biology, and reproductive immunology. The results of the research are also relevant to dairy farmers, cattle ranchers, and embryo technology practitioners. Efforts will be made at the appropriate time to convey results and recommendations for changes in reproductive management and embryo production to these groups. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In the project period, research contributed to the doctoral research programs of three students, the masters research program of one student, and to the research program of an intern from Brazil. How have the results been disseminated to communities of interest?Seminar: Exploitation of developmental programming in the preimplantation period to enhance fertility and improve postnatal function in cattle. Zoetis, Kalamazoo, MI, November 4, 2015. What do you plan to do during the next reporting period to accomplish the goals?Finish an experiment to evaluate whether CSF2 treatment from day 5-7 after insemination alters the characteristics of the fetus and placenta at Day 90 of gestation.
Impacts
What was accomplished under these goals?
Colony stimulating factor 2 can have multiple effects on the function of the preimplantation embryo that include increased potential to develop to the blastocyst stage, reduced apoptosis, and enhanced ability of inner cell mass (ICM) to remain pluripotent after culture. Recently, we identified genes regulated by CSF2 in the ICM and trophectoderm (TE) of the bovine blastocyst with the goal of identifying possiblemolecular pathways by which CSF2 increases developmental competence for survival. Embryos were produced in vitro and cultured from Day 6 to 8 in serum-free medium containing 10 ng/ml recombinant bovine CSF2 or vehicle.Blastocysts were harvested at Day 8 and ICM separated from TE by magnetic-activated cell sorting. RNA was purified and used to prepare amplified cDNA, which was then subjected to high-throughput sequencing using the SOLiD 4.0system. Three pools of amplified cDNA were analyzed per treatment. The number of genes whose expression was regulated by CSF2, using P < 0.05 and >1.5-fold difference ascut-offs, was 945 in the ICM (242 upregulated by CSF2 and 703 downregulated) and 886 in the TE (401 upregulated by CSF2 and 485 downregulated). Only 49 genes were regulated in a similar manner by CSF2 in both cell types. The three significant annotation clusters in which genes regulated by ICM were overrepresented were related to membrane signaling. Genes downregulated by CSF2 in ICM were overrepresented in several pathways including those for ERK and AKT signaling. The only significant annotation cluster containing an overrepresentation of genes regulated byCSF2 in TE was for secreted or extracellular proteins. In addition, genes downregulated in TE were overrepresented in TGFβ and Nanog pathways. Conclusions: Differentiation of the blastocyst is such that, by Day 8 after fertilization, the ICM and TE respond differently to CSF2. Analysis of the genes regulated by CSF2 in ICM and TE are suggestive that CSF2 reinforces developmental fate and function of both cell lineages. In vitro production (IVP) of embryos can disrupt fetal and placental development and increase risk of abnormal fetal growth. Maternal factors play a role in developmental programming of the early embryo. Colony-stimulating factor 2 (CSF2) is present in the oviduct and endometrium and has improved competence of the preimplantation embryo to establish pregnancy in cattle. Another experiment was performed with the objective of determiningwhether CSF2 during embryo culture alters fetal development and alleviates abnormalities associated with IVP. Holstein oocytes were matured and fertilized in vitro with X-sorted semen from a Holstein bull. Putative zygotes were cultured in SOF-BE1 at 5% CO2 and 5% O2 for 5 days and then randomly assigned to receive vehicle (IVP-control) or 10 ng/mL CSF2 (IVP-CSF2). Grade I blastocysts were transferred on day 7 to Holstein recipients that were previously randomized to receive an IVP-control or an IVP-CSF2 embryo. A third group of cows included in the randomization was assigned to be artificially inseminated on day 0 using the same bull as for IVP (AI). Pregnancy was terminated on day 85 or 86. Statistical analysis was performed by analysis of variance using the GLM procedure of SAS with contrasts for AI vs (IVP-control+IVP-CSF2) (contrast 1; C1) and IVP-control vs IVP-CSF2 (contrast 2; C2). A total of 23 morphometric measurements of placenta and fetus were made on 9 AI, 12 IVP and 7 CSF2 female singletons. Conceptuses derived by IVP (IVP-control and IVP-CSF2) differed from those derived by AI for four characteristics including fetal body weight (142.9 ± 4.7, 157.2 ± 4.4, and 162.6 ± 6.1 g for AI, IVP-control and IVP-CSF2, respectively; C1, P=0.0237), eviscerated weight (102.9 ± 3.4, 113.6 ± 3.2, and 112.2 ± 4.4 g; C1, P=0.0602), crown-rump length (CRL) (13.7 ± 0.2, 14.0 ± 0.2, and 14.7 ± 0.3 g; C1, P=0.0434; C2, P=0.0631) and umbilical cord diameter (0.85 ± 0.08, 1.1 ± 0.08, and 0.91 ± 0.1 cm; P=0.0519). Note that while IVP-CSF2 conceptuses were generally similar to those for IVP-control, CRL tended to be highest for IVP-CSF2. Also, umbilical cord diameter for IVP-CSF2 was similar to AI and lower than IVP-control. Data from one fetus in the IVP-CSF2 group was excluded from analysis because it had a phenotype consistent with large offspring syndrome. Body weight (354 g) was two-fold larger than other fetuses (average = 155 g) and placental weight was seven-fold greater (1,505 vs 211 g). In addition, organs were enlarged and severe ascites and hemorrhagic cotyledons were observed. In conclusion, IVP resulted in increased fetal size and umbilical cord diameter without other significant effects on placental morphometry. CSF2 did not alleviate adverse effects of culture on fetal growth, exacerbating effects on CRL, but did reduce effects of IVP on umbilical cord diameter. Gene expression analysis in fetal liver, muscle, and placenta is underway tofurther characterizeeffects of CSF2.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Denicol, A.C., Le�o, B.C.S., Dobbs, K.B., Mingoti, G.Z., and Hansen, P.J. (2015) Influence of sex on basal and dickkopf-1 regulated gene expression in the bovine morula. Plos One 10, e0133587.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Kannampuzha-Francis, J., Denicol, A.C., Loureiro, B., Kaniyamattam, K., Ortega, M.S., and Hansen, P.J. (2015) Exposure to colony stimulating factor 2 during preimplantation development increases postnatal growth in cattle. Mol. Reprod. Dev. 82, 892-897.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Ozawa, M., Sakatani, M., Dobbs, K.B., Kannampuzha-Francis, J., and Hansen, P.J. (2016) Regulation of gene expression in the bovine blastocyst by colony stimulating factor 2. BMC Res. Notes 9, 250.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Chen, Z., Hagen, D.E., Wang, J., Elsik, C.G., Tieming, J., Siqueira, L.G., Hansen, P.J., and Rivera, R.M. (2016) Global assessment of imprinted gene expression in the bovine conceptus by next generation sequencing. Epigenetics 11, 501-516.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2016
Citation:
Kannamphuza-Francis, J., Tribulo, P., and Hansen, P.J. (2016) Actions of activin A, connective tissue growth factor, hepatocyte growth factor and teratocarcinoma-derived growth factor 1 on development of the bovine embryo. Reprod. Fertil. Dev., in press.
|
Progress 09/01/14 to 08/31/15
Outputs
Target Audience: The primary target audience are research scientists with a focus on embryology, reproductive biology, pregnancy biology, stem cell biology, and reproductive immunology. The results of the research are also relevant to dairy farmers, cattle ranchers, and embryo technology practitioners. Efforts will be made at the appropriate time to convey results and recommendations for changes in reproductive management and embryo production to these groups. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? In the project period, research contributed to the doctoral research programs of three students, the masters research program of one student, and to the research program of an intern from Brazil. How have the results been disseminated to communities of interest? Seminars: Developmental programming of the preimplantation embryo by colony stimulating factor 2 - a case of male vs female. AJRI Award Lecture, 34th Annual Meeting of the American Society for Reproductive Immunology, Long Beach, NY, June 2-5, 2014. Consequences of interactions between the maternal immune system and the preimplantation embryo in cattle. 9th International Ruminant Reproduction Symposium, Obihiro, Japan, August 25-29, 2014. SSR Exchange Lecture: Colony stimulating factor 2: Example of a maternal cytokine that programs the mammalian embryo for pregnancy success. American Society for Reproductive Medicine 47th Annual Postgraduate Program, October 18-22, 2014, Honolulu, Hawaii. Colony stimulating factor 2 and Dickkopf 1 - Maternal embryokines that program the bovine embryo for pregnancy success. Dept. of Animal and Poultry Sciences, Virginia Polytechnic & State University, Blacksburg, VA, November 21, 2014. Maternal embryokines that program the bovine embryo for pregnancy success. H. Allen and Ann Tucker Lectureship Seminar Series, Michigan State University, March 23, 2015. What do you plan to do during the next reporting period to accomplish the goals? Finish an experiment to evaluate whether CSF2 treatment from day 5-7 after insemination alters the characteristics of the fetus and placenta at Day 90 of gestation. Evaluate consequences of exposure to CSF2 during the preimplantation period for postnatal growth.
Impacts
What was accomplished under these goals?
Recently, we have shown that CSF2 can alter the pattern of embryonic development in a sex-dependent manner in the cow. Exposure of embryos to CSF2 during Day 5-7 of development, when the embryo is undergoing the morula-blastocyst transition, has pronounced and sex-dependent effects on characteristics of embryos later in pregnancy when the embryo is undergoing a rapid elongation of the trophoblast required for maintenance of the corpus luteum (34). Briefly, in vitro produced embryos were treated with either 0 or 10 ng/ml bovine CSF2 from Day 5 to 7. Expanded blastocysts were transferred into synchronized recipients and collected on Day 15. Sex was determined by PCR and data obtained for 6-9 embryos for each sex-treatment combination. There was a sex x treatment interaction (P<0.05) for embryo length and secretion of the antiluteolytic IFNT in uterine flushing. CSF2 increased embryo length and IFNT secretion in males and decreased length and IFNT in females (Figure 2). Analysis of the transcriptome of extra-embryonic membranes using the Agilent EmbryoGENE bovine microarray also indicated that sex affected the response to CSF2. Treatment caused differential regulation of 94 genes in males and 56 genes in females. Only 7 differentially-regulated genes were regulated in both sexes and, when shared, regulation by CSF2 was always in the opposite direction. The EmbryoGENE DNA Methylation Array was used to assess CSF2 effects on methylation status of extraembryonic membranes at 418,805 CpG located across the genome. CSF2 caused hypermethylation for 9,842 probes in males and 6,227 probes in females. Hypomethylation was detected for 9,322 probes in males and 3292 probes in females. Only 1186 probes were regulated by CSF2 in both sexes, and regulation was in the opposite direction for 772 of these probes. Developmental plasticity in response to maternal environment is an evolutionary strategy which allows individuals to adapt to the environment during their life, thereby preserving the genotype in the face of transient environmental changes). One of the characteristics of developmental programming in both lizards and mammals is that postnatal phenotype can depend on sex. Sexual dimorphism in developmental programming may reflect the differential benefit of altered adult phenotype to males and females as well as differences in evolutionary importance of males and females for reproductive success in various environments. Our recent results suggests that sexual dimorphism in developmental programming of mammals during preimplantation development is dictated by disparate actions of maternally-derived factors on male and female embryos.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Hansen, P.J., Denicol, A.C., and Dobbs, K.B. (2014) Maternal embryokines that regulate development of the bovine preimplantation embryo. Turkish J. Anim. Vet. Sci. 38, 589-598.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Hansen, P.J., Dobbs, K.D., and Denicol, A.C. (2014) Programming of the preimplantation embryo by the embryokine colony stimulating factor 2. Anim. Reprod. Sci. 149, 59-66.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Dobbs, K.B., Gagn�, D., Fournier, E., Dufort, I., Robert, C., Block, J., Sirard, M.-A., Bonilla, L., Ealy, A.D., Loureiro, B., and Hansen, P.J. (2014). Sexual dimorphism in developmental programming of the bovine preimplantation embryo caused by colony stimulating factor 2. Biol. Reprod. 91, 80.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Denicol, A.C., Block, J., Kelley, D.E., Pohler, K.G., Dobbs, K.B., Mortensen, C.J., Ortega, M.S., and Hansen, P.J. (2014) The WNT signaling antagonist Dickkopf-1 directs lineage commitment and promotes survival of the preimplantation embryo. FASEB J. 28, 3975-3986.
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Hansen, P.J. (2014). Consequences of interactions between the maternal immune system and the preimplantation embryo in cattle. In: Juengel, J.L., Miyamoto, A., Reynolds, L.P., Smith, M.F., and Webb, R., eds., Reproduction in Domestic Ruminants VIII. Context Publishing, Packington Leicestershire, pp 141-152.
|
Progress 09/01/13 to 08/31/14
Outputs
Target Audience: The primary target audience are research scientists with a focus on embryology, reproductive biology, pregnancy biology, stem cell biology, and reproductive immunology. The results of the research are also relevant to dairy farmers, cattle ranchers, and embryo technology practitioners. Efforts will be made at the appropriate time to convey results and recommendations for changes in reproductive management and embryo production to these groups. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Five graduate students have contributed to the project as well as a visiting graduate student from UNESP in Brazil and two undergraduate students. How have the results been disseminated to communities of interest? Data were presented to bovine practitioners at the XIX Congreso Asociación Nacional de Especialistas en Medicina Bovina de España, Oviedo, Spain, June 25-27, 2014. What do you plan to do during the next reporting period to accomplish the goals? -determine changes in the transcriptome and methylome caused by CSF2 in fetal and placental tissues at Day 90 of gestation -determine the mechanism of action by which CSF2 signals to embryonic cells
Impacts
What was accomplished under these goals?
Some of the changes in gene expression in the embryo caused by CSF2 are indicative that CSF2 blocks WNT signaling. Accordingly, a series of experiments were conducted to determine whether dickkopf-1, an modulator of WNT signaling that blocks canonical WNT signaling, would alter embryo cell-fate decisions and competence to establish pregnancy. Exposure of embryos to DKK1 during the period of morula-to-blastocyst transition (between Days 5 and 8 of development) promoted the first two cell-fate decisions leading to increased differentiation of cells towards the trophectoderm and hypoblast lineages as compared to control embryos treated with vehicle. Moreover, treatment of embryos with DKK1 or CSF2 between Days 5 and 7 of development improves embryo survival following transfer to recipients. Pregnancy success at Day 32 of gestation was 27% for cows receiving control embryos treated with vehicle, 41% for cows receiving embryos treated with DKK1 and 39% for cows receiving embryos treated with CSF2. These novel findings represent the first evidence for a role for maternally-derived WNT regulators during this period and could lead to improvements in assisted reproductive technologies. The fact that DKK1 affects lineage committment differently than does CSF2 (which increases numbers of inner cell mass cells without affecting trophectoderm cell number) indicates that DKK1 is exerting actions on the embryo to promote its survival that are distinct from those of CSF2.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
248. Dobbs, K.D., Khan, F.A., Sakatani, M., Moss, J.I., Ozawa, M., Ealy, A.D., and Hansen, P.J. (2013) Regulation of pluripotency of inner cell mass and growth and differentiation of trophectoderm of the bovine embryo by colony stimulating factor 2. Biol. Reprod. 89, 141.
|
Progress 09/01/12 to 08/31/13
Outputs
Target Audience: The primary target audience are research scientists with a focus on embryology, reproductive biology, pregnancy biology, stem cell biology, and reproductive immunology. The results of the research are also relevant to dairy farmers, cattle ranchers, and embryo technology practitioners.Efforts will be made at the appropriate time to convey results and recommendations for changes in reproductive management and embryo production to these groups. Changes/Problems: We are extending our research to include actions of the WNT inhibitorDKK1 on embryonic development because of data that CSF2 causes changes in the transcriptome that could lead to adownregulation of canonical WNT signaling. What opportunities for training and professional development has the project provided? Three graduate students have contributed to the project as well as a visiting graduate student from UNESP in Brazil and two undergraduate students. How have the results been disseminated to communities of interest? Results of some of the research were reported to embryo transfer practitioners at the First International Embryo Transfer Society Innovation Workshop held in St. Augustine Florida in October 2012. What do you plan to do during the next reporting period to accomplish the goals? -continue to evaluate changes in the methylome in Day 15 embryos caused by CSF2 -determine changes in the transcriptoe of Day 15 embryos and relate these changes to the changes in the methylome -determine changes in the transcriptome and methylome caused by CSF2 in fetal and placental tissues at Day 90 of gestation -determine the mechanism of action by which CSF2 signals to embryonic cells
Impacts
What was accomplished under these goals?
Three experiments were conducted to determine the mechanism by which treatment of bovine embryos with CSF2 from Day 5-7 post-insemination alters the developmental program of the embryo to increase initial pregnancy rate and reduce late embryonic and fetal loss. In one experiment, the hypothesis was tested that regulates pluripotency of the inner cell mass (ICM) and growth and lineage commitment of the trophectoderm (TE). Embryos produced in vitro were cultured from day 5-7 or day 6-8 post-insemination (pi) with either 10 ng/ml recombinant bovine CSF2 or vehicle. ICM isolated immunosurgically from blastocysts produced in the presence of CSF2 were more likely to survive passage when cultured on mitomycin-C treated fetal fibroblasts in the presence of the GSKB inhibitor CHIR99021 and MEK1inhibitor PD0325901 than ICM from control blastocysts. Surviving ICM colonies expressed the pluripotency marker NANOG . Actions of CSF2 to increase survival were not due to the anti-apoptotic actions of CSF2, since a pan-caspase inhibitor tended to reduce survival of ICM, or to regulation of expression of NANOG, SOX2, CDX2 or GATA6, since steady-state concentrations of transcripts for these genes in the ICM were not affected by CSF2. In contrast to effects on ICM, characteristics of TE outgrowth from day 7 or 8 blastocysts were generally unaffected by CSF2 except that expression of CDX2 was increased 1.4-fold by CSF2 from day 6-8 pi. (1.43-fold). In a second experiment, it was tested whether CSF2 changes the degree of methylation of the ICM and TE of bovine embryos. Using immunofluorescent labeling with anti-5-methylcytosine, it was demonstrated that methylation decreased from the 2-cell stage to the 6-8 cell stage and then increased thereafter up to the blastocyst stage. Methylation was greater for female embryos at the 8-cell stage but greater for males at the blastocyst stage. Treatment with CSF2 had no effect on labeling for DNA methylation in blastocysts. Methylation was lower for inner cell mass cells (i.e., cells that did not label with anti-CDX2) than for trophectoderm (CDX2-positive). Thus, there appears to be no large scale change in the methylome caused by CSF2. To evaluate consequences of CSF2 action on development at later stages of pregnancy, we evaluated whether treatment with CSF2 from Day 5-7 alters growth, interferon-τ (IFNT) secretion, and the methylome of extra-embryonic membranes (EEM) at Day 15. In vitro produced embryos were treated with either 0 or 10 ng/ml bovine CSF2 from Day 5 to 7. Expanded blastocysts were transferred into synchronized recipients. On Day 15, conceptuses were recovered by flushing the uterus and gender determined by PCR (n=4 males and 4 females per treatment). There was a gender x treatment interaction for conceptus length (P<0.003) and IFNT in uterine flushing (P<0.05) (as determined by antiviral activity). CSF2 increased length in males and decreased length in females. Similarly, CSF2 increased IFNT in males and decreased IFNT in females. The EmbryoGENE DNA Methylation Array was used to assess CSF2 effects at 418,805 positions across the genome in a subset of EEM. CSF2 caused hypermethylation for 9,842 probes in males and 6,227 probes in females and hypomethylation for 9,322 probes in males and 3,292 probes in females. An analysis was conducted to evaluate if clusters of differentially-methylated probes were non-randomly distributed spatially in 5 Mb regions of individual chromosomes using a 500 kb moving window. Differential methylation was not uniformly distributed but rather there were regions or hyper- and hypomethylation that varied with gender. The most common situation was where a region was differentially methylated in one gender but not the other. In some cases, a region was differentially methylated in opposite directions for males and females. For example, a region of BTA 1 between positions 67Mb-77.5Mb contained 1,632 probes of which 59 probes were hypo-methylated in males and 35 were hypermethylated in females. These results indicate changes in developmental programming of the bovine embryo caused by CSF2 occur in a gender-specific manner. This result suggests a possible mechanism by which environmental effects on the female affect male embryos differently than female embryos. Finally, an experiment was conducted to determine whether, like in the mouse, the preimplantation bovine embryo only expresses one of the two CSF2 receptor subunit genes, expression of CSF2RA and CSFRB was determined. Expression of CSF2RA increased from the zygote stage to the 9-16 cell stage before decreasing to the blastocyst stage. In contrast, CSF2RB was undetectable at all stages.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
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. (2012) Global gene expression of the inner cell and trophectoderm of the bovine blastocyst. BMC Dev. Biol. 12, 33
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Denicol, A.C., Dobbs, K.B., McLean, K.M., Carambula, S.F., Loureiro, B., and Hansen, P.J. (2013) Canonical WNT signaling regulates development of bovine embryos to the blastocyst stage. Sci. Rep. 3,1266
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Dobbs, K.B., Rodriquez M., Sudano, M.J., Ortega, M.S. and Hansen, P.J. (2013) Dynamics of DNA methylation during early development of the preimplantation bovine embryo. PLoS ONE 8, e66230
|
Progress 09/01/11 to 08/31/12
Outputs
OUTPUTS: The first distinct differentiation event in mammals occurs at the blastocyst stage when totipotent blastomeres differentiate into either pluripotent inner cell mass (ICM) or multipotent trophectoderm (TE). Here we determined, for the first time, global gene expression patterns in the ICM and TE isolated from bovine blastocysts. In addition, we evaluated how CSF2, which increases embryo competence to complete development to term, alters gene expression. Bovine embryos were produced in vitro and were treated with 0 or 10 ng/ml CSF2 from Day 6 to 8 after insemination. The ICM and TE were isolated by magnetic activated cell sorting, and cDNA sequenced using the SOLiD 4.0 system. A total of 870 genes were differentially expressed between ICM and TE. CSF2 changed expression of 25 genes in ICM and 23 in TE. Several genes characteristic of ICM (for example, NANOG, SOX2, and STAT3) and TE (ELF5, GATA3, and KRT18) in mouse and human showed similar patterns in bovine. Other genes, however, showed differences in expression between ICM and TE that deviates from the expected based on mouse and human. PARTICIPANTS: PJ Hansen Manabu Ozawa (postdoctoral training) Miki Sakatani (postdoctoral training) Kyle Dobbs (doing PhD) TARGET AUDIENCES: the main target audience is the scientific community. The work is submitted for publication and has been presented at a sympoisum on Functional Genomics of Livestock Embryos in Banff, Canada. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Analysis of gene expression indicated that differentiation of blastomeres of the morula-stage embryo into the ICM and TE of the blastocyst is accompanied by differences between the two cell lineages in expression of genes controlling metabolic processes, endocytosis, hatching from the zona pellucida, paracrine and endocrine signaling with the mother, and genes supporting the changes in cellular architecture, stemness, and hematopoiesis necessary for development of the trophoblast. Specific genes regulated by CSF2 may be targets for improving fertility.
Publications
- No publications reported this period
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