Progress 01/15/12 to 01/14/16
Outputs Target Audience:As the research supported by this grant is fundamental-basic research, the primary audience is other scientists conducting research in reproductive biology. Accordingly, research funded by this grant was presented this past year at the following venues: Altering specific functions of the ruminant placenta: New approaches to assess causation of fetal growth restriction. March 28, 2015. Experimental Biology meetings/American Society of Nutrition Symposia "Maternal/Fetal Nutrition and Programming: What Have We Learned from Farm Animal Models?," Boston, MA. Research supported by this grant was presented at the Annual Meeting of the Society for the Study of Reproduction during 2015, and will also be presented at the Annual Meeting of the Society for the Study of Reproduction this comming summer (2016) in San Diego. Additionally, the PI organizes the Front Range Pregnancy Consortium (FRPC) which was held June 5 and October 30, 2015 in Fort Collins, CO, where the results of this research were discussed. The FRPC had attendees from Colorado State University, University of Colorado-Anshutz Medical Center and the University of Wyoming. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Callie M. Baker was a M.S. student supported by this grant, and the research reported provided the basis for her M.S. Thesis. Callie graduated in December 2014. Lindsey N. Goetzmann, who served as a Research Associate in Dr. Anthony's laboratory following completion of her undergraduate research in Dr. Anthony's laboratory, was supported by this project before entering an Physicians Assistant program in January, 2014. She was subsequently replaced by Dr. Kimberly Jeckel, Research Scholar 1, who has continued the research efforts supported by this grant. How have the results been disseminated to communities of interest?As the research supported by this grant is fundamental-basic research, the primary audience is other scientists conducting research in reproductive biology. Accordingly, research funded by this grant was presented this past year at the following venue: Altering specific functions of the ruminant placenta: New approaches to assess causation of fetal growth restriction. March 28, 2015. Experimental Biology meetings/American Society of Nutrition Symposia "Maternal/Fetal Nutrition and Programming: What Have We Learned from Farm Animal Models?," Boston, MA. Research supported by this grant was presented at the Annual Meeting of the Society for the Study of Reproduction during 2015, and will also be presented at the Annual Meeting of the Society for the Study of Reproduction this comming summer (2016) in San Diego. Additionally, the PI organizes the Front Range Pregnancy Consortium (FRPC) which was held June 5 and October 30, 2015 in Fort Collins, CO, where the results of this research were discussed. The FRPC had attendees from Colorado State University, University of Colorado-Anshutz Medical Center and the University of Wyoming. Finally, the results from the 135 dGA pregnancies was recently published in the American Journal of Physiology. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts What was accomplished under these goals?
IMPACT: While our knowledge of fetal growth regulation in livestock is limited, undoubtedly the placenta plays a major role in determining fetal growth rate. This is obvious from its role as the interface between the maternal and fetal systems, providing for nutrient transport to the fetus and waste removal from the fetus. Additionally, the placenta is a rich source of hormones and growth factors that likely impact the fetus, mother or both. The existence of the placental lactogens(PLs) has been known for over 50 years, and while it has been hypothesized for some time that human and sheep PL play an active role in modulating maternal and fetal metabolism, this hypothesis has never been directly assessed in vivo. We have developed methods by which we can inhibit expression of specific genes within the placenta in vivo in sheep. During the past year, we have continued to analyse the tissue harvested from the 135 dGA pregnancies (Objective 3), as well as tissues derived from the 50 dGA pregnancies (Objective 1 part 2). We did not generate and assess pregnancies at 90 dGA as originally planned, as we felt it necessary to repeat Objective 1 with only singleton pregnancies, using the most efficacious lentiviral constructs (Lentilox 3.7 expressing tg6 or a scrambled control sequence). Significant growth restriction has now been demonstrated at both gestational ages, and is likely mediated by oPLs effect on stimulating the insulin-like growth factors (IGFs), which in turn may well alter glucose transporter (SLC2A1 and SLC2A3) expression within the placenta. The impact of this basic research is three-fold. First, it demonstrates that we can use this placenta-specific transgenic technology to create a deficiency in a very abundant hormone produced by the placenta (oPL). Second, it demonstrates that PL does indeed play an important role during pregnancy, impacting fetal development. Third, it demonstrates the PL may also have an important role in regulating placental development. In brief, we have been successful in generating sheep pregnancies that have significant fetal and placental growth restriction by specifically altering the expression of a single gene within the placenta. This validates a new approach for examining gene function during pregnancy in livestock, allowing future assessment of specific factors that may regulate the outcome of pregnancy, and ultimately impact postnatal growth and development of livestock. Specific Outcomes: Using the lentiviral-mediated transgenesis approaches that we developed, we have generated oPL deficient pregnancies which were examined at two gestation ages. At 50 dGA, a 41% reduction (P<0.05) in uterine vein oPL was achieved, and was associated with a 20.5% (P<0.05) reduction in fetal weight, a 20.9% reduction (P<0.01) in fetal liver weight, yet only a 16.5% (P=0.11) reduction in placental weight. In contrast to the major effect that oPL deficiency had on fetal liver insulin-like growth factor (IGF) expession at 135 dGA (see below), expression of IGF1 and IGF2 expression were only reduced 17% and 29% respectively, at 50 dGA, with no significant effects on IGF binding protein (IGFBP) expression. At 50 dGA, while fetal weight was signficantly reduced, placental weight only tended to be, yet placental expression of IGF1 and IGF2 mRNA were both reduced 53%, and IGFBP2 mRNA concentration was reduced 73% (P<0.05) with no effect on the other IGFBPs. In addition, placental SLC2A1 mRNA concentration was reduced 43% (P<0.05) at 50 dGA, whereas SLC2A3 mRNA concentration was not significantly reduced. As reported in our recent manuscript in the American Journal of Physiology, oPL deficiency resulted in a 32% reduction in fetal weight and a 52% reduction in placental weight, accompanied by a 40% reduction in fetal liver weight (all P<0.01), was observed at 135 dGA. Fetal liver IGF1 and IGF2 mRNA concentrations were reduced (P<0.01) 82% and 71%, respectively, and umbilical artery IGF1 concentrations were reduced 62% (P<0.01) in oPL deficient pregnancies. Additionally, fetal liver IGFBP2 and oIGFBP3 mRNA concentrations were reduced (P<0.05), whereas fetal liver oIGFBP1 mRNA concentration was not impacted nor was maternal liver IGF and IGFBP mRNA concentrations or uterine artery IGF1 concentrations (P>0.10). Accompaning the reduction in placental weights were a 65% reduction in IGF1, a 53% reduction in IGF2, a 55% reduction in SLC2A1 and a 49% reduction in SLC2A3 mRNA concentration (all P<0.05). It has long been hypothesized that PL functioned to modulate maternal and fetal amino acid, carbohydrate and lipid metabolism, possibly by stimulating IGF synthesis and secretion. Our data support this concept, and while not initially hypothesized, it appears that PL promotes placental growth and function, as well as fetal growth, likely through its impact on IGF production. Furthermore, our data support the concept that PL stimulates IGF production, which in turn stimulates the expression of at least SLC2A1 and possibly SLC2A3, the placental transporters involved in transfer of glucose to the fetus. Expression of SLC2A1 appears to be more impacted by PL deficiency and the associated decline in IGF1 expression, and that SLC2A3's role may be greatest during late gestation when fetal growth is maximal.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2016
Citation:
Baker, C.M. L.N. Goetzmann, J.D. Cantlon, K.M. Jeckel, Q.A. Winger and R.V. Anthony. 2016. The development of ovine chorionic somatomammotropin hormone deficient pregnancies. Am. J. Physiol.- Reg. Integrat. and Comp. Physiol. e-published 2/17/16.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Romero, J.J., A.Q. Antoniazzi, T.M. Nett, R.L. Ashley, B.T. Webb, N.P. Smirnova, R.C. Bott, J.E. Bruemmer, F.W. Bazer, R.V. Anthony, and T.R. Hansen. 2015. Temporal release, paracrine and endocrine actions of ovine conceptus-derived interferon-tau during early pregnancy. Biol. Reprod. 93:146, 1-10.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2016
Citation:
Barry, J.S., P.J. Rozance, L.D. Brown, R.V. Anthony, K.L.Thornburg and W.W Hay, Jr. 2016. Increased fetal myocardial sensitivity to insulin-stimulated glucose metabolism during ovine fetal growth restriction. Exp. Biol. Med. e-published 2/11/16.
|
Progress 01/15/14 to 01/14/15
Outputs Target Audience: As the research supported by this grant is fundamental-basic research, the primary audience is other scientists conducting research in reproductive biology. Accordingly, research funded by this grant was presented this past year at the following venues: Assessing gene function in the placenta. Feb. 11, 2014. Program in Reproductive Sciences, Department of Obstetrics and Gynecology, University of Colorado-Denver and Health Sciences Center, Aurora, CO; Altering specific functions of the ruminant placenta: New approaches to assess causation of fetal growth restriction. March 28, 2015. Experimental Biology meetings/American Society of Nutrition Symposia "Maternal/Fetal Nutrition and Programming: What Have We Learned from Farm Animal Models?," Boston, MA; July 19, 2014, Annual USDA-NIFA-AFRI Animal Reproduction Investigators meeting, Grand Rapids, MI; July 20, 2014, Development of a placental lactogen deficient pregnancy in sheep. 47th Annual Society for the Study of Reproduction meeting, Grand Rapids, MI. Additionally, the PI initiated the Front Range Pregnancy Consortium (FRPC) which was held September 19, 2014 in Fort Collins, CO, where the results of this research were discussed. The FRPC had attendees from Colorado State University, University of Colorado-Anshutz Medical Center and the University of Wyoming. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Callie M. Baker was a M.S. student is supported by this grant, and the research reported will provided the basis for her M.S. Thesis. Callie graduated in December 2014. Lindsey N. Goetzmann, who served as a Research Associate in Dr. Anthony's laboratory following completion of her undergraduate research in Dr. Anthony's laboratory, was supported by this project before entering an Physicians Assistant program in January, 2014. She was subsequently replaced by Dr. Kimberly Jeckel, Research Scholar 1, who is completing the research supported by this grant. How have the results been disseminated to communities of interest? Research funded by this grant was presented this past year at the following venues: Assessing gene function in the placenta. Feb. 11, 2014. Program in Reproductive Sciences, Department of Obstetrics and Gynecology, University of Colorado-Denver and Health Sciences Center, Aurora, CO; Altering specific functions of the ruminant placenta: New approaches to assess causation of fetal growth restriction. March 28, 2015. Experimental Biology meetings/American Society of Nutrition Symposia "Maternal/Fetal Nutrition and Programming: What Have We Learned from Farm Animal Models?," Boston, MA; July 19, 2014, Annual USDA-NIFA-AFRI Animal Reproduction Investigators meeting, Grand Rapids, MI; July 20, 2014, Development of a placental lactogen deficient pregnancy in sheep. 47th Annual Society for the Study of Reproduction meeting, Grand Rapids, MI. What do you plan to do during the next reporting period to accomplish the goals? We will finish the sample analysis during this year of no-cost extension. In addition, we are generating CRISPR/Cas9 expression constructs in lentivirus, with the goal of introducing mutations in the oPL gene in vivo as another approach to generate PL deficient pregnancies. This is a new technological approach which has yet to be applied to large animal placenta. The potential advantage is that it will introduce mutations in the gene, rather than rely on continue targeting of oPL mRNA throughout gestation. Somatic cell nucelar transfer as well as direct zygote microinjection of CRISPR/Cas9 constructs has been used in large animals, but our goal is to obtain placenta specific mutations. While PL is only produced by the placenta, many other genes of interest are not placenta specfic, such that "complete" knockout will could directly impact fetal development, making assessment of specific actions difficult at best. That is why we have chosen to continue with our lentiviral targerting of the trophectoderm cells.
Impacts What was accomplished under these goals?
IMPACT: While our knowledge of fetal growth regulation in livestock is limited, undoubtedly the placenta plays a major role in determining fetal growth rate. This is obvious from its role as the interface between the maternal and fetal systems, providing for nutrient transport to the fetus and waste removal from the fetus. Additionally, the placenta is a rich source of hormones and growth factors that likely impact the fetus, mother or both. The existence of the placental lactogens(PLs) has been known for over 50 years, and while it has been hypothesized for some time that human and sheep PL play anactive role in modulating maternal and fetal metabolism, this hypothesis has never been directly assessed in vivo. We have developed methods by which we can inhibit expression of specific genes within the placenta in vivo in sheep. During the past year we completed the stated objectives for Objective 3, that being to complete the horomone and tissue analysis for the singleton pregnancies harvested at 135 days of gestation. Because we had observed such a significant effect on placental weight (52% reduction) and fetal weight (32%) reduction at 135 dGA, to determine how early these effects might be initiated we generated additional pregnancies that were harvested at 50 dGA. At 50 dGA we observed a significant reduction (22%) in fetal weight. The impact of this basic research is three-fold. First, it demonstrates that we can use this placenta-specific transgenic technology to create a deficiency in a very abundant hormone produced by the placenta (oPL). Second, it demonstrates that PL does indeed play an important role during pregnancy, impacting fetal development. Third, it demonstrates the PL may also have an important role in regulating placental development. In brief, we have been successful in generating sheep pregnancies that have significant fetal and placental growth restriction by specifically altering the expression of a single gene within the placenta. This validates a new approach for examining gene function during pregnancy in livestock, allowing future assessment of specific factors that may regulate the outcome of pregnancy, and ultimately impact postnatal growth and development of livestock. Objective 3: The 52% reduction in placental mass was associated with a 50% reduction in oPL mRNA and a 38% reduction in placental oPL concentration. While fetal weight was reduced 32%, fetal liver weights were reduced 41% (P≤0.05), yet fetal liver insulin-like growth factor (IGF)-1 and -2 mRNA concentrations were reduced (P≤0.01) 82% and 71%, respectively, and a 62% reduction in umbilical artery IGF-1 concentration. There was a similar reduction in IGFBP-2 (74%) and IGFBP-3 (81%) mRNA, without an effect on IGFBP-1 expression. In contrast, maternal liver expression of IGF-1, IGF-2, IGFBP-1, IGFBP-2 and IGFBP-3 mRNA was not impacted, nor was maternal uterine artery concentration of IGF-1. Maternal and fetal glucose concentrations were not altered in these pregnancies, although there was a 35% increase in the uterine artery-uterine vein glucose gradient, suggesting greater placental glucose uptake. While maternal insulin concentrations were not impacted, there was a 49% decline in umbilical artery insulin concentration. Objective 1 (part 2): When we originally conducted Objective 1, the results were confounded by the fact that we transferred multiple blastocysts to each recipeint, as we were only interested in determining the knockdown efficiency. Base on the results from Objective 3, we wanted to determine how early these effects would occur during gestation. To this end we transferred single-infected blastocysts into synchronized ewes and harvested the pregnancies at 50 dGA. Fetal weights and fetal liver weights were resuced 22 and 21% respectively, whereas crown-rump lenght was reduced 11%, with a non-significan reduction in placental weight (17%). In cotrast to the 135 dGA data, fetal liver IGF-1 and IGF-2 mRNA concentrations were only reduce 17% and 29%, respectively. However, day 50 placental IGF-1 and IGF-2 mRNA concentraitons were reduced 52% and 51%, respectively. While tissue and hormone analysis from these pregnancies has just begun, it is apparent that early reductions in PL production impacts early fetal growth. It has long been hypothesized that PL functioned to modulate maternal and fetal amino acid, carbohydrate and lipid metabolism, possibly by stimulatin inuslin like growth factor synthesis and secretion. Our data support this concept, and while not initially hypothesized, it appears that PL promotes placental growth and function, as well as fetal growth.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Cleys, E.R., J.L. Halleran, V.A. Enriquez, J.C. da Silveira, R.C. West, Q.A. Winger, R.V. Anthony, J.E. Bruemmer, C.M. Clay and G.J. Bouma. 2015. Androgen receptor and histone lysine demethylases in ovine placenta. PLoS One 10(2):e0117472.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Baker, C.M., L.N. Goetzmann, J.D. Cantlon, Q.A. Winger and R.V. Anthony. 2014. Development of a placental lactogen deficient pregnancy in sheep. 47th Annual Society for the Study of Reproduction meeting. Abstract 340.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Keller-Wood, M, X. Feng, C.E. Wood, E. Richards1, R.V. Anthony, G.E. Dahl and S. Tao. 2014. Elevated maternal cortisol leads to relative maternal hyperglycemia and increased stillbirth in ovine pregnancy. Am. J. Physiol.- Reg, Integrat. and Comp. Physiol. 307: R405-413.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Field, M.E., R.V. Anthony, T.E. Engle, S.L. Archibeque, D.H. Keisler and H. Han. 2015. Duration of maternal undernutrition differentially alters fetal growth and hormone concentrations. Dom. Anim. Endocrinol. 51:1-7.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Henkes, L.E., J.K. Pru, R.L. Ashley, R.V. Anthony, D.N.R. Veeramachaneni, K.C. Gates and T.R. Hansen. 2015. Embryo Mortality in Isg15-/- mice is Exacerbated by Environmental Stress. Biol. Reprod. 92(2):36, 110
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Progress 01/15/13 to 01/14/14
Outputs Target Audience: As the research supported by this grant is fundamental-basic research, the primary audience is other scientists conducting research in reproductive biology. Accordingly, research funded by this grant was presented this past year at the following three venues. Molecular Control of Pre- and Peri-Attachment Conceptus Development. March 21, 2013. 60th Society for Gynecologic Investigation Annual Meeting. "New Insights into the Contribution of the Mother to Embryogenesis and Placentation in Early Pregnancy" Symposium, Orlando, FL. Progression of Ovine Placental Lactogen Deficient Pregnancies. July 26, 2013. Annual USDA-NIFA-AFRI Animal Reproduction Investigators meeting, Montreal, Canada. Assessing Gene Function in the Ruminant Placenta. August 26, 2013. Aspen/Snowmass Perinatal Biology Symposium "Fetal Adaptation to Maternal and Placental Dysfunction," Snowmass Village, CO. Changes/Problems: We plan A Change in Action for this upcoming year. Since we have already conducted Objective 3, and demonstrated that creating placental lactogen deficiency resulted in significant fetal and placental growth restriction, we plan to use our lentiviral methodology to accomplish two things. The first is to "overexpress" oPL throughout gestation by infecting blastocysts with a hEF-1α-oPL expression construct that we have generated and tested in vitro. In vitro, this construct was very effective, such that its expression by all trophoblast cells, not just the chorionic binucleate cells, should result in "excessive" oPL which may create larger placenta at 90 days of gestation (selected gestational endpoint). We base the hypothesis of greater placental growth on our results demonstrating greater placental growth restriction than fetal growth restriction in our oPL deficient pregnancies. The second change is to generate CRISPR/Cas9 expression constructs in lentivirus, with the goal of introducing mutations in the oPL gene in vivo as another approach to generate PL deficient pregnancies. This is a new technological approach which has yet to be applied to large animals and specifically the placenta. The potential advantage of this approach is that it will introduce mutrations in the gene, rather than rely on continued targting of oPL mRNA throughout gestation. These pregnancies will be allowed to go to 90 days of gestation before harvest. One concern is that if we are able to more robustly reduce oPL early on in pregnancy, these pregnancies may in fact be lost before 90 days of gestation. All pregnancies will be assessed by serial transabdominal ultrasound assessment of pregnancy status, and if pregnancy demise begins to occur, we will harvest the pregnancies earlier. If successful with this approach, this will provide another transgenic technology for use in livestock to assess specific gene function. What opportunities for training and professional development has the project provided? Callie M. Baker is a M.S. student who is supported by this grant, and the research reported will provide the basis for her M.S. Thesis. Rachel West, a new PhD student started her program in January, 2014, and is involved in this research, but it is not the focus of her doctoral research. Lindsey N. Goetzmann, who served as a Research Associate in Dr. Anthony's laboratory following completion of her undergraduate research in Dr. Anthony's laboratory, was supported by this project before entering an Physicians Assistant program in January, 2014. How have the results been disseminated to communities of interest? During the past year, results from these studies were presented at two international scientific meetins, through invited lectures by Dr. Anthony, as well as at the annual USDA-NIFA-AFRI investigator meeting. Molecular Control of Pre- and Peri-Attachment Conceptus Development. March 21, 2013. 60th Society for Gynecologic Investigation Annual Meeting. "New Insights into the Contribution of the Mother to Embryogenesis and Placentation in Early Pregnancy" Symposium, Orlando, FL. Progression of Ovine Placental Lactogen Deficient Pregnancies. July 26, 2013. Annual USDA-NIFA-AFRI Animal Reproduction Investigators meeting, Montreal, Canada. Assessing Gene Function in the Ruminant Placenta. August 26, 2013. Aspen/Snowmass Perinatal Biology Symposium "Fetal Adaptation to Maternal and Placental Dysfunction," Snowmass Village, CO. What do you plan to do during the next reporting period to accomplish the goals? We plan A Change in Action for this upcoming year. Since we have already conducted Objective 3, and demonstrated that creating placental lactogen deficiency resulted in significant fetal and placental growth restriction, we plan to use our lentiviral methodology to accomplish two things. The first is to "overexpress" oPL throughout gestation by infecting blastocysts with a hEF-1α-oPL expression construct that we have generated and tested in vitro. In vitro, this construct was very effective, such that its expression by all trophoblast cells, not just the chorionic binucleate cells, should result in "excessive" oPL which may create larger placenta at 90 days of gestation (selected gestational endpoint). We base the hypothesis of greater placental growth on our results demonstrating greater placental growth restriction than fetal growth restriction in our oPL deficient pregnancies. The second change is to generate CRISPR/Cas9 expression constructs in lentivirus, with the goal of introducing mutations in the oPL gene in vivo as another approach to generate PL deficient pregnancies. This is a new technological approach which has yet to be applied to large animals and specifically the placenta. The potential advantage of this approach is that it will introduce mutrations in the gene, rather than rely on continued targting of oPL mRNA throughout gestation. These pregnancies will be allowed to go to 90 days of gestation before harvest. One concern is that if we are able to more robustly reduce oPL early on in pregnancy, these pregnancies may in fact be lost before 90 days of gestation. All pregnancies will be assessed by serial transabdominal ultrasound assessment of pregnancy status, and if pregnancy demise begins to occur, we will harvest the pregnancies earlier. If successful with this approach, this will provide another transgenic technology for use in livestock to assess specific gene function.
Impacts What was accomplished under these goals?
IMPACT: While our knowledge of fetal growth regulation in livestock is limited, undoubtedly the placenta plays a major role in determining fetal growth rate. This is obvious from its role as the interface between the maternal and fetal systems, providing for nutrient transport to the fetus and waste removal from the fetus. Additionally, the placenta is a rich source of hormones and growth factors that likely impact the fetus, mother or both. The existence of the placental lactogens(PLs) has been known for over 50 years, and while it has been hypothesized for some time that human and sheep PL play anactive role in modulating maternal and fetal metabolism, this hypothesis has never been directly assessed in vivo. We have developed methods by which we can inhibit expression of specific genes within the placenta in vivo in sheep. During the past year we completed our analysis of Objective 1, that being to generate and test the efficacy of three different lentiviral ovine PL (oPL) targeting vectors at 50 days of gestation. From this we determined that we obtained a 66% average reduction of oPL secretion from the placenta into maternal blood. Subsequently, we used three lentiviral oPL targeting vectors, and a control vector, to generate singleton-transgenic pregnancies which were harvested recently at 135 days of gestation (near-term). While all three of the targeting vectors worked, two provided an average of 30% fetal growth restriction and 48% placental growth restriction. The impact of this basic research is three-fold. First, it demonstrates that we can use this placenta-specific transgenic technology to create a deficiency in a very abundant hormone produced by the placenta (oPL). Second, it demonstrates that PL does indeed play an important role during pregnancy, impacting fetal development. Third, it demonstrates the PL may also have an important role in regulating placental development. In brief, we have been successful in generating sheep pregnancies that have significant fetal and placental growth restriction by specifically altering the expression of a single gene within the placenta. This validates a new approach for examining gene function during pregnancy in livestock, allowing future assessment of specific factors that may regulate the outcome of pregnancy, and ultimately impact postnatal growth and development of livestock. Objective 1: Objective 1 was completed with the assay of oPL concentrations in the uterine vein (UtV) draining each pregnancy. Based on a 95% confidence interval created from UtV oPL concentrations ipsilateral to the fetus (n=6) in naturally-mated control (NMC) pregnancies, 4 out of 7 hLL3.7-oPL-tg6 placenta, 5 out of 7 hEF-1-oPLtg2-shRNAmiR placenta and 5 out of 7 oPGK-oPLtg2-shRNAmiR placenta were classified as responders. Compared to NMC (41.8±3.58 ng/ml), UtV oPL concentrations, 16.8±5.00 ng/ml (hLL3.7-oPL-tg6), 12.48±1.36 ng/ml (hEF-1-oPLtg2-shRNAmiR) and 13.75±2.29 ng/ml (oPGK-oPLtg2-shRNAmiR) were reduced (P≤0.01), representing 59.7%, 70.1% and 67.1% reduction in oPL secretion in these pregnancies, respectively. While we hypothesize that oPL deficiency will result in IUGR near-term, at 55 dGA there were no differences in fetal weights. With the degree of UtV oPL concentration reductions, we decided to move forward with three targeting vectors for our future work, and to bypass our assessment at 90 days of gestation (Objective 2). Consequently, we decided to shift to Objective 3 for the 2013-14 breeding season, rather than Obective 2. This preliminary study supported the concept that production of an abundant hormone, PL, could be effectively diminished using this transgenic technology. Objective 3: Day 9 hatched and expanded blastocysts were harvested from naturally-mated donor ewes, and then infected for 5 hours with either hEF-1α-SC, hEF-1α-oPL shRNAmiR 2, hEF-1α-oPL shRNAmiR 6 or hLL3.7-oPL-tg6 before surgical transfer of single blastocysts to synchronized recipient ewes. the hEF-1α-SC vector incorporates the human EF-1α promoter driving the expression of a scramble control (SC) shRNAmiR (microRNA mimic). Both hEF-1α-oPL shRNAmiR 2 and hEF-1α-oPL shRNAmiR 6 use the human EF-1α promoter driving the expression of oPL target 2 or target 6 as shRNAmiRs built within miR-30. For hLL3.7-oPL-tg6, we replaced the mouse U6 (RNA Pol III) promoter with the human U6 promoter to drive the expression of oPL-target 6 in the form of a short-hairpin RNA (shRNA). Seventy-six percent of the single blastocysts transferred on day 9 resulted in pregnancies, as assessed by transabdominal ultrasound after 40 days of gestation (dGA). At 135 dGA, 8 hEF-1α-SC pregnancies, 9 hEF-1α-oPL shRNAmiR 2 pregnancies, 7 hEF-1α-oPL shRNAmiR 6 pregnancies and 9 hLL3.7-oPL-tg6 pregancies were harvested. At pregnancy harvest, catheters were placed into the umbilical artery (UmbA) and vein (UmbV), as well as the uterine artery (UtA) and vein (UtV) ipsilateral to the fetus to obtain simultaneous blood sampling. Following blood sampling, the fetus was euthanized and removed, at which point the ewe was euthanized and the uterus and liver were removed. Fetal weights and crown-rump lengths were determined, and the fetal liver was weighed and snap frozen for later analysis. All of the placentomes were removed from the uterus, counted, weighed (placental weight), and intact placentomes were sectioned and processed for immunohistochemistry, or dissected into maternal caruncular or fetal cotyledonary tissue, and the fetal cotyledons were snap frozed for analysis. Ninety-five percent confidence intervals were calculated for fetal weights and placenta weights obtained from the hEF-1α-SC control pregnancies, and hEF-1α-oPL shRNAmiR 2, hEF-1α-oPL shRNAmiR 6 and hLL3.7-oPL-tg6 pregnancies that had both placental and fetal weights that were less that the 95% confidence interval were considered to be responders. Based on this classification, which could change once the blood and tissue analysis is complete, 4 out of 9 (44%) hEF-1α-oPL shRNAmiR 2 pregnancies, 3 out of 7 (43%) hEF-1α-oPL shRNAmiR 6 pregnancies and 6 out of 9 (67%) hLL3.7-oPL-tg6 pregancies were classified as responders. Placental weights, fetal weights, fetal liver weights and fetal crown-rump lengths for the responders were statistically compared to hEF-1α-SC control pregnancies by Student's T-test. Fetal weights were less (P<0.01)in hEF-1α-oPL shRNAmiR 2 (3.94±0.22 kg; 20% reduction) pregnancies, hEF-1α-oPL shRNAmiR 6 (3.23±0.42 kg; 33% reduction) and hLL3.7-oPL-tg6 (3.46±0.44 kg; 29% reduction) when compared to hEF-1α-SC control pregnancies (4.90±0.11 kg). Placenta were smaller (P<0.01) in hEF-1α-oPL shRNAmiR 2 (0.58±0.07 kg; 26% reduction) pregnancies, hEF-1α-oPL shRNAmiR 6 (0.41±0.14 kg; 48% reduction) and hLL3.7-oPL-tg6 (0.40±0.04 kg; 48% reduction) when compared to hEF-1α-SC control pregnancies (0.78±0.03 kg). Similarily, fetal crown-rump lengths and fetal liver weights were also significantly reduced in the hEF-1α-oPL shRNAmiR 2, hEF-1α-oPL shRNAmiR 6 and hLL3.7-oPL-tg6 pregnancies. However, fetal ponderal index was not impacted, suggesting that the fetal growth restriction that ensued in these pregnancies were symmetrical in nature, typically associated with an early onset of fetal growth restriction. While hormone analysis and tissue analysis from these pregnancies has just begun, it is clear that targeting oPL expression early in pregnancy had a major impact on the progression of placental and fetal growth. Additionally, it appears that our hLL3.7 vector is more efficacious for these long-term studies than the more complex "second generation" vectors.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Peel, R.K., G.J. Eckerle and R.V. Anthony. 2012. Effects of overfeeding naturally-mated adolescent ewes on maternal, fetal and postnatal lamb growth. J. Anim. Sci. 90:3698-3708.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Seabrook, J.L., J.D. Cantlon, A.J. Cooney, E.E. McWhorter, B.A. Fromme, G.J. Bouma, R.V. Anthony and Q.A. Winger. 2013. Role of Lin28A in mouse and human trophoblast cell differentiation.
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Progress 01/15/12 to 01/14/13
Outputs OUTPUTS: Objective 1 of this project was to generate and test the efficacy of different lentiviral vectors designed to knockdown the expression of ovine placental lactogen (oPL) both in vitro as well as in vivo at 55 days of gestation (dGA) in sheep. CHO, Cos-7 and Ach-3P cells were stably transfected with an oPL over-expression vector (pcDNA3). These cell lines were then used to test the in vitro efficiency of oPL mRNA "knockdown" as a result of our lentiviral oPL targeting vectors. Two oPL-specific targeting sequences were tested, and are designated as "target 2" and "target 6." Both targeting sequences were used to generate lentiviral vectors, using two different strategies. The first utilized the human U6 promoter to drive the expression of target 2 or target 6 as simple short-hairpin RNA (shRNA), and are designated as LL3.7-t6 or LL3.7-t2. The second utilized the cytomegalovirus (CMV), elongation factor-1 (EF1) ubiquitin C (UBC) or the phosphoglycerate kinase (PGK) to drive the expression of oPL-specific shRNA embedded within miR30, designated as CMV-oPLtg6-shRNAmiR, CMV-oPLtg2-shRNAmiR, EF1-oPLtg6-shRNAmiR, EF-1-oPLtg2-shRNAmiR, UBC-oPLtg6-shRNAmiR, UBC-oPLtg2-shRNAmiR, PGK-oPLtg6-shRNAmiR and PGK-oPLtg2-shRNAmiR. While all of the targeting vectors were effective in significantly reducing the expression of oPL in our cell lines, the LL3.7-tg6, EF-1-oPLtg2-shRNAmiR and PGK-oPLtg6-shRNAmiR were the most effective, providing greater than 95% knockdown across all of the cell lines. The CMV promoter was the least effective, especially within the human trophoblast cell line Ach-3P, only providing 40-70% loss of oPL mRNA. Accordingly, LL3.7-tg6, EF1-oPLtg2-shRNAmiR and PGK-oPLtg6-shRNAmiR were used to infect day 8 hatched blastocysts, followed by surgical transfer to synchronized recipient ewes. The recipient ewes, along with naturally-mated control ewes were allowed to gestate until 55 dGA, when uterine vein blood was collected for oPL radioimmunoassay, along with the fetus (weight and crown-rump length) and placenta (mRNA and protein concentrations) for analysis. Of the healthy blastocysts infected and transferred on day 8, at 55 dGA 88% of those blastocysts resulted in a viable fetus/placenta. As the harvest of the 55 dGA samples were just recently completed, the analysis of the samples is ongoing. The results obtained from Objective 1 will determine which lentiviral construct we use to assess the impact of oPL deficiency at 90 dGA (Objective 2) and 135 dGA (Objective 3). PARTICIPANTS: Dr. Russell V. Anthony is the PD of this project, and participates in and oversees all aspects of this project. Jeremy Cantlon is a Research Associate in Dr. Anthony's laboratory and is involved in all aspects of this project. Callie M. Baker is a Masters of Science student in the Department of Biomedical Sciences, who is supported by this grant, and her M.S. thesis will be derived from this research. Vincent Abushaban is an undergraduate student in the Department of Animal Sciences student, and is supported by this grant and assists with the management, breeding, etc. of our sheep, and is being trained in laboratory research techniques in support of this research project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts During Year 1, we were able to establish several cell lines that over-expressed ovine placental lactogen (oPL), which were then used to assess the effectiveness of a number of lentiviral constructs to target the degradation of oPL mRNA in vitro. While all of the lentiviral oPL-targeting constructs were effective in vitro, we identified much greater variability in their effectiveness when tested within a trophoblast cell line (Ach-3P). This was primarily true for the CMV-oPL-shRNAmiR constructs, which varied between 40% and 70% oPL mRNA loss, whereas the constructs that utilized the EF1, UBC or PGK promoters provided more consistent results across the cell lines. Through these studies, and others, we are left to conclude that CMV, while a strong promoter, is less robust in trophoblast cells and becomes extinguished over time, making it an unsuitable promoter for use in our in vivo studies. Consequently, one EF1 (EF1-oPLtg2-shRNAmiR) and one PGK (PGK-oPL-tg6-shRNAmiR) construct were chosen for use in vivo, along with LL3.7-t6. We chose to use LL3.7-t6 as it contains a RNA Polymerase III promoter (U6) driving the expression of a simple shRNA, rather than a shRNAmiR, and this targeting strategy worked very well for our previous studies on Proline-Rich 15 (PRR15; Purcell et al., 2009, Biol. Reprod.) in the sheep conceptus. While we have recently finished harvesting the 55 dGA pregnancies, and sample analysis is ongoing, our preliminary results are providing insight as how to move forward with this research. First, for the healthy blastocysts transferred following lentiviral infection, 88% of those resulted in viable fetuses/placenta. This is consistent with our previous studies, and could suggest that the viral infection actually enhances the survival of these blastocyst following surgical transfer. Regardless of the cause, in the future we will transfer only one healthy blastocyst/recipient, such that only singleton pregnancies will be studied, increasing the statistical power of the results. Second, preliminary analysis of a subset of the samples suggests that the amount of reduction in oPL mRNA within the 55 dGA placenta was not as great as observed in the cell lines, but there is a greater reduction in the amount of oPL (as assessed by Western blot analysis) then inferred by mRNA concentrations and that this is being born out by the radioimmunoassay measurement of oPL concentrations in uterine vein blood. If the complete analysis of all of the samples collected demonstrate the same thing, this would infer that in vitro we obtain primarily mRNA degradation, but when this approach is applied in vivo that we get a combination of shRNA or shRNAmiR driven mRNA degradation along with inhibition of mRNA translation. While these results are preliminary at best, we are encouraged that we will be able to go forward with Objectives 2 and 3, with the aim of testing the hypothesis that oPL deficiency in vivo will result in intrauterine growth restriction of the fetus.
Publications
- No publications reported this period
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