Progress 05/15/17 to 05/14/21
Outputs
Target Audience:The primary audience is the community of scientists involved in animal reproduction. Other audiences arethe pharmaceutical industry, veterinary practitioners, livestock advisors, and dairy farmers. Changes/Problems:Objective 1 was modified to change the focus from measuring gene expression changes caused by DKK1 to further understanding how DKK1 and other WNT signals modify characteristics of the embryo and to identify signaling pathways regulated by DKK1. The change was made because the new objectives were deemed more important for gaining an understanding of DKK1 biology. What opportunities for training and professional development has the project provided?Two graduate students and a postdoc conducted the research for the project. Thiago Amaral, working on a PhD in Animal Molecular and Cellular Biology,carried out most of the work described in this report. Surawich Jeensuk performed studies with WNT5A. She is a MS student in Animal Molecular and Cellular Biology. Experiments on embryonic stem cells were performed by Yao Xiao, who has now taken a faculty position in China. How have the results been disseminated to communities of interest?The following presentations have been given to scientific and veterinary groups. Programming of development during embryonic and fetal life in cattle and its consequences for subsequent production. Theriogenology Conference 2018, Society for Theriogenology, Milwaukee, WI, August 1-4, 2018 Developmental programming of development during the preimplantation period: Examples from the cow. Women and Infants Hospital, Brown University, November 28 2018. Developmental programming in the preimplantation period - the phenomenon and prospects for control. Zoetis, Kalamazoo, MI, December 10, 2018. Maternal control of embryo competence for pregnancy success - the role of embryokines. Annual Meeting of the American Dairy Science Association, Cincinatti, OH, June 23-27, 2019. Developmental programming during the preimplantation period - impact of the environment of the preimplantation embryo on characteristics of the progeny. 13th Simposio Internacional de Reproduccion Animal, Cordoba, Argentina, Aug 28-30, 2019. Embryo transfer - a promising tool for improving fertility during heat stress. 35th Annual Meeting, Asocciation for Embryo Technology in Europe, Murcia, Spain, Sept 13-14, 2019. Maternal control of embryo competence for pregnancy success. Faculty of Veterinary Medicine, University of Murcia, September 16, 2019. Maternal control of embryo competence for pregnancy success - the role of embryokines. Dept. of Animal Science, North Carolina State University, February 12, 2020. Utilizing embryo transfer as a way of mitigating the effects of heat stress on reproduction. 2020 Joint Virtual Convention, American Embryo Transfer Assn/Canadian Embryo Tansfer Assn., October 6-7, 2020. Embryokines and maternal control of embryo competence for pregnancy success. Save the Egg Webinar Series, Colorado State University, June 16 2020. What does a good embryo look like? Molecular markers of competence to establish pregnancy after transfer. Séerie Conhecimenta Científico, Universidade Federal do Pampa, July 30 2020. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
DKK1 is an inhibitor of WNT signaling, which in turn is an important cell signaling system that plays important roles in development and organogenesis. Preliminary experiments that lead to the current project indicated that DKK1 is produced by the endometrium of the cow and can act on the blastocyst to increase the number of trophectoderm cells (giving rise to the placenta),increasecompetence of embryos to establish pregnancy and program fetal development to reduce birth weight. These results suggested it is important to inhibit WNT signaling in the preimplantation embryo and that doing so might be one approach for increasing pregnancy rate in cattle and for programming development of the calf to improve postnatal function. The first objective (measure gene expression caused by DKK1) was modified. The new focus was on further characterizinghow DKK1 affects differentiation and pluripotency of the cells of the embryo and determine some of the signalingmechanism by which DKK1 modifies embryo function.The work (which has not yet been published) indicates that DKK1 can indeed increase the number of trophectoderm cells. We also showed that pharmacological inhibition of WNT signaling can maintain embryonic stem cells derived from blastocysts in a primed pluripotent state (i.e., capable of differentiating into several but not all cell types) but that DKK1 does not have the same effect. Thus, inhibition of WNT signaling is important to maintain cells of the embryo in a pluripotent state but this effectcannot be achieved by DKK1 alone. It was also shown that mRNA for the DKK1 receptor, KREMEN1, is expressed by the embryo and that expression declines as the embryo advances in development. Preliminary results (the experiments are ongoing)also indicate that the KREMEN1 protein is present in the embryo as is an alternate receptor called CKAP4. As expected, DKK1 reduces WNT signaling as indicated by a reduction in the amounts of beta-catenin, the second messenger for canonical WNT. We also examined whether otherWNT molecules can regulatethe embryo. The WNT tested, WNT5A, often exhibits actions in other cells that are opposed to those of otherWNT. We found thatWNT5A can increase the percent of embryos becoming blastocysts and the number of cells in the inner cell mass (the part of the embryo that gives rise to the fetus). Given the fact that DKK1 increases the number of trophectoderm cells, it is possible that one function of DKK1 is to enhance elongation of the trophoblast at day 15 of pregnancy. In cattle, this elongation is necessary for the embryo to signal its presence to the mother and allow pregnancy to continue. Signaling is mediated by a trophoblast-derived protein called interferon-tau. For Objective 2, an experiment was conducted in which embryos were treated with DKK1 or control from day 5 to 7 of gestation,transferred into recipient females, and then flushed from the uterus at Day 15 of pregnancy. As hypothesized, DKK1 caused the embryos to be more elongated and to produce more interferon-tau. These results support the idea that DKK1 may improve pregnancy rate by enhancing signaling by interferon-tau. Recent experiments from our laboratory indicate that signaling molecules that affect the embryo in the preimplantation period can modify the course of development in a way that changes fetal development and the characteristics of the offspring. For Objective 3, we conducted three experiments using a total of 2770 cows to evaluate whether DKK1 treatment of embryos from day 5 to 7 of development would increase pregnancy rate and change the phenotype of the resultant calves. Additional embryos were treated with colony stimulating factor 2 (CSF2), because this molecule has also been shown to affect pregnancy rate and calf phenotype. Treatment with DKK1 did not improve pregnancy rate. Treatment with CSF2 improved pregnancy rate when embryos were cultured in a serum-containing medium but decreased pregnancy rate when embryos were in a medium without serum. The experiments are still ongoing so that data on the calves born from the project can be evaluated. We obtained additional evidence for theidea that maternal signals can alter fetal development for Objective 3. In particular, the methylome of fetal muscle and liver was examined for embryos produced in vivo,in vitro or in vitro in the presence of CSF2, which has been shown to affect embryonic development. There were differences in specific methylated regions of the DNA between the three treatments. Thus, one mechanism by which maternal signals act to program the fetus is likely through alterations of epigenetic control of gene expression. Overall, we learned important information about the role of WNT signaling, and especially the WNT regulator DKK1, in regulation of the early embryo. Preliminary results indicating that DKK1 might be a useful treatment to increase pregnancy rate were not supported by new experiments. Whether DKK1 affects the characteristics of the resultant calves will be revealed towards the autumn of 2021.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Hansen PJ. (2020) The incompletely fulfilled promise of embryo transfer in cattle-why aren't pregnancy rates greater and what can we do about it? J. Anim Sci. 98, skaa288.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Li, Y., Tr�bulo, P., Bakhtiarizadeh, M.R., Siqueira, L.G., Ji, T., Rivera, R.M., and Hansen, P.J. (2020) Conditions of embryo culture from days 5 to 7 of development alter the DNA methylome of the bovine fetus at day 86 of gestation. J. Assist. Reprod. Genet. 37, 417-426.
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Progress 05/15/19 to 05/14/20
Outputs
Target Audience:The primary audience is the community of scientists involved in animal reproduction. Other audiences are the pharmaceutical industry, veterinary practitioners, livestock advisors, and dairy farmers. Changes/Problems:Objective 1 was modified to change the focus from measuring gene expression changes caused by DKK1 to further understanding how DKK1 and other WNT signals modify characteristics of the embryo and to identify signaling pathways regulated by DKK1. The change was made because the new objectives were deemed more important for gaining an understanding of DKK1 biology. What opportunities for training and professional development has the project provided?A graduate student, Thiago Amaral, working on a PhD in Animal Molecular and Cellular Biology, carried out most of the work described in this report. Experiments on embryonic stem cells were performed by Yao Xiao, who has now taken a faculty position in China How have the results been disseminated to communities of interest?Maternal control of embryo competence for pregnancy success - the role of embryokines. Dept. of Animal Science, North Carolina State University, February 12, 2020. Utilizing embryo transfer as a way of mitigating the effects of heat stress on reproduction. 2020 Joint Virtual Convention, American Embryo Transfer Assn/Canadian Embryo Tansfer Assn., October 6-7, 2020. Embryokines and maternal control of embryo competence for pregnancy success. Save the Egg Webinar Series, Colorado State University, June 16 2020. What does a good embryo look like? Molecular markers of competence to establish pregnancy after transfer. Séerie Conhecimenta Científico, Universidade Federal do Pampa, July 30 2020. What do you plan to do during the next reporting period to accomplish the goals?The grant period is finished but we still have a great deal of data to collect and prepare for publication.
Impacts
What was accomplished under these goals?
Recent experiments indicate that signaling molecules that affect the embryo in the preimplantation period can modify the course of development in a way that changes fetal development and the characteristics of the offspring. For Objective 3, we conducted three experiments using a total of 2770 cows to evaluate whether DKK1 treatment of embryos from day 5 to 7 of development would increase pregnancy rate and change the phenotype of the resultant calves. Additional embryos were treated with colony stimulating factor 2 (CSF2), because this molecule has also been shown to affect pregnancy rate and calf phenotype. Treatment with DKK1 did not improve pregnancy rate. Treatment with CSF2 improved pregnancy rate when embryos were cultured in a serum-containing medium but decreased pregnancy rate when embryos were in a medium without serum. The experiments are still ongoing so that data on the calves born from the project can be evaluated.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Hansen PJ. (2020) The incompletely fulfilled promise of embryo transfer in cattle-why aren't pregnancy rates greater and
what can we do about it? J. Anim Sci. 98, skaa288.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Li, Y., Tr�bulo, P., Bakhtiarizadeh, M.R., Siqueira, L.G., Ji, T., Rivera, R.M., and Hansen, P.J. (2020) Conditions of embryo
culture from days 5 to 7 of development alter the DNA methylome of the bovine fetus at day 86 of gestation. J. Assist.
Reprod. Genet. 37, 417-426.
|
Progress 05/15/18 to 05/14/19
Outputs
Target Audience:The primary audience is the community of scientists involved in animal reproduction. Other audiences are thepharmaceutical industry, veterinary practitioners, livestock advisors, and dairy farmers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Thiago Amaral performed research on the project that is contributing towards his doctoral dissertation. How have the results been disseminated to communities of interest?Two seminars on the research were presented as follows: Developmental programming of development during the preimplantation period: Examples from the cow. Women and Infants Hospital, Brown University, November 28 2018. Developmental programming in the preimplantation period - the phenomenon and prospects for control. Zoetis, Kalamazoo, MI, December 10, 2018. What do you plan to do during the next reporting period to accomplish the goals?We will continue studies to characterize how DKK1 affects the transcriptome of the blastocyst, evaluate the cell signaling pathway by which DKK1 regulates the preimplantation embryo, and conduct additional experiments to evaluate whether treatment of the preimplantation embryo with DKK1 alters competence of the embryo to establish pregnancy and postnatal characteristics of the resultant calf.
Impacts
What was accomplished under these goals?
Progesterone regulates the endometrium to support pregnancy establishment and maintenance. In theruminant, one action of progesterone early in pregnancy is to alter embryonic development and hastenthe process of trophoblast elongation around day 14-15 of pregnancy, which is required for maternalrecognition of pregnancy. We tested whether the WNT antagonist DKK1, whose expression isincreased by progesterone treatment, can act on the bovine embryo during day 5 to 7.5 of development(the morula to blastocyst stage) to promote embryonic elongation on day 15 of pregnancy. Embryoswere produced in vitro and exposed to 0 or 100 ng/ml recombinant human DKK1 from day 5 to 7.5 ofculture. Blastocysts were transferred into synchronized recipient cows on day 7.5 (n = 23 for control and 17 for DKK1). On day 15, cows were slaughtered and embryos recovered by flushing the uterus.Embryo recovery was n = 11 for controls (48% recovery) and n = 11 for DKK1 (65% recovery). Exceptfor two DKK1 embryos, all embryos were filamentous. Treatment with DKK1 increased (P = 0.007) thelength of filamentous embryos from 43.9 mm to 117.4 mm and the intrauterine content of the maternal recognition of pregnancy signal IFNT (P = 0.01) from 4.9 μg to 16.6 μg. Determination of differentiallyexpressed genes (DEG), using the R environment, revealed 473 DEG at p < 0.05 but none at FDR < 0.05,suggesting that DKK1 did not strongly modify the embryo transcriptome at the time it was measured.However, samples clustered apart in a multidimensional scaling analyisis. Weighted gene co-expressionanalysis of the transcriptome of filamentous embryos revealed a subset of genes that were relatedto embryo length, with identification of a significant module of genes in the DKK1 group only. Thus,several of the differences between DKK1 and control groups in gene expression were due to differencesin embryo length. In conclusion, DKK1 can act on the morula-to-blastocyst stage embryo to modifysubsequent trophoblast elongation. Higher pregnancy rates associated with transfer of DKK1-treatedembryos may be due in part to enhancements of trophoblast growth and antiluteolytic signalingthrough IFNT secretion. Given that progesterone can regulate both timing of trophoblast elongationand DKK1 expression, DKK1 may be a mediator of progesterone effects on embryonic development.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
303. Tr�bulo P, Rabaglino MB, Bo MB, Carvalheira LR, Bishop JV, Hansen TR, Hansen PJ. (2019) Dickkopf-related protein 1 is a progestomedin acting on the bovine embryo during the morula-to-blastocyst transition to program trophoblast elongation. Sci Rep. 9, 11816.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
53. Hansen, P.J., and Tr�bulo, P. (2019) Regulation of present and future development by maternal regulatory signals acting on the embryo during the morula to blastocyst transition - insights from the cow. Biol Reprod. 101, 526-537.
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Progress 05/15/17 to 05/14/18
Outputs
Target Audience:The primary audience is the community of scientists involved in animal reproduction. Other audiences are the pharmaceutical industry, veterinary practitioners, livestock advisors, and dairy farmers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Paula Tribulo conducted research for the project as a postdoctoral scientist. How have the results been disseminated to communities of interest?Data have been published in the scientific literature. In addition, results have been reported to embryo transfer practioners, veterinarians via invited talks at the following venues: Postnatal consequences of reproductive technologies in cattle. 2017 American Embryo Transfer Assn./Canadian Embryo Transfer Assn. Joint Annual Convention, Orlando, Florida, October 26-28, 2017. Programming of development during embryonic and fetal life in cattle and its consequences for subsequent production. Theriogenology Conference 2018, Society for Theriogenology, Milwaukee, WI, August 1-4, 2018. What do you plan to do during the next reporting period to accomplish the goals?Evaluate effect of DKK1 on elongation of the embryo at day 15 of pregnancy; initiate studies to test whether DKK1 can change the transcriptome of the bovine blsatocyst
Impacts
What was accomplished under these goals?
Embryokines are molecules secreted by the mother that regulate embryonic development. Among these molecules in cattle are colony stimulating factor 2 (CSF2) and dickkopf-related protein 1 (DKK1). Here, we evaluated actions of CSF2 and DKK1 alone or in combination on characteristics of embryos produced in vitro in the presence of serum. A total of 70 beef cows from 4 farms were subjected to oocyte retrieval on 1 to 4 occasions. Within each farm, donors were randomly allocated to 1 of 4 treatment groups (vehicle, CSF2, DKK1, CSF2 + DKK1). Embryos from a given donor were always exposed to the same treatment. Treatments were added to the culture medium on d 5 after insemination, and blastocyst stage embryos were transferred to recipient females 2 d later. Treatment did not affect the percent of oocytes or cleaved embryos that developed to the blastocyst stage or the percent of recipients that became pregnant afterembryo transfer. However, calves derived from embryos treated with DKK1 were smaller at birth, regardless of CSF2 treatment. Results indicate no effects of addition of CSF2 or DKK1 to culture of embryos produced in vitro with serum-containing medium on development to the blastocyst stage or competence to establish pregnancy after transfer to recipients. The fact that embryos cultured with DKK1 resulted in calves with reduced birth weight illustrates the potential ability of thisembryokine to program postnatal phenotype. Results support the concept that properties of the offspring can be programmed as early as the preimplantation period.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Consequences of exposure of embryos produced in vitro in a serum-containing medium to dickkopf-related protein 1 and colony stimulating factor 2 on blastocyst yield, pregnancy rate, and birth weight
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