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Browsing Theses and Dissertations by Author "Anthony, Russell V., advisor"

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    Characterization of periattachment factor
    (Colorado State University. Libraries, 2008) Purcell, Scott H., author; Anthony, Russell V., advisor; Seidel, George E., advisor
    The purpose of these studies was to characterize expression of periattachment factor (PF) mRNA in the developing sheep conceptus, localize PF in the sheep conceptus, determine if degrading PF mRNA in the sheep conceptus was detrimental to development, and characterize PF in human tissues and cells. Sheep conceptuses were collected on d 11, 13, 15, 16, 17, 21, and 30 post-mating. oPF mRNA exhibited a quadratic expression pattern (P<0.05). No oPF mRNA was detected in d 11 conceptuses. From d 13, oPF mRNA increased to a peak at d 16 before declining. Peak expression of oPF mRNA in the conceptus occurs during rapid elongation and initial attachment of the conceptus to the endometrium. Immunolocalization of PF in a d 15 conceptus showed predominantly nuclear staining in trophectoderm and trophendoderm. Next, embryos collected from superovulated ewes on d 8 post-mating were infected with either a lentivirus expressing a short-hairpin (sh) RNA designed to target PF mRNA for degradation, a lentivirus expressing a shRNA containing 3 mismatched nucleotides, or a control lentivirus expressing no shRNA. Following infection, blastocysts were transferred into recipient ewes and collected back on d 15 of gestation. While 94 and 88% of the control and mismatched shRNA-treated conceptuses elongated by d 15, none of the embryos treated with the lentivirus expressing shRNA against PF mRNA elongated, and most died. This indicates that oPF is required for conceptus elongation and survival. Human PF mRNA was detected in human carcinomas and in the first trimester cytotrophoblast cell line, hTR8. Immunohistochemistry showed PF in the nuclei of carcinomas and in first and second trimester cytotrophoblasts. PF was also present in the invading cytotrophoblast columns. In an in vitro invasion assay of first trimester cytotrophoblasts, hPF mRNA increased from 0, 3, to 12 h as invasion occurred. To further characterize PF in human cells, a lentiviral construct expressing an shRNA targeting the hPF mRNA sequence was developed that resulted in 63% mRNA reduction in BeWo cells.
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    The development of ovine placental lactogen deficient pregnancies
    (Colorado State University. Libraries, 2014) Baker, Callie M., author; Anthony, Russell V., advisor; Winger, Quinton A., committee member; Curthoys, Norman, committee member
    Intrauterine growth restriction (IUGR) results in significant fetal and neonatal mortalities and morbidities. Additionally, infants surviving IUGR experience increased incidence of heart disease, diabetes, hypertension and stroke during adulthood. A major placental secretory product, placental lactogen (PL), is found at high levels in maternal and fetal circulation, and is significantly reduced in both human and sheep IUGR pregnancies. While the exact function of PL has not been defined for any species, it is thought to modulate the mobilization of maternal nutrients to the fetus. Recently, the development of lentiviral-mediated expression of short hairpin RNA (shRNA) within sheep conceptuses has provided a means of examining placental gene function in sheep. The objective of this research was to generate ovine PL (oPL) deficient sheep pregnancies using lentivirus targeting the degradation of oPL mRNA, in order to assess the function of PL during pregnancy. We hypothesize that oPL deficiency during pregnancy will lead to IUGR near term. To test our hypothesis a preliminary study was conducted to evaluate the in vivo efficacy of lentiviral-oPL targeting vectors in the sheep placenta at 55 days gestational age (dGA). Efficiency of oPLmRNA degradation was first measured in vitro using twelve promoter-targeting sequence combinations, tested in three cell lines overexpressing oPL. Two oPL target sequences (tg2 and tg6) were used, as either shRNA or shRNAmiR (microRNA mimic) sequences. Subsequently, three lentiviral constructs; hLL3.7 tg6 (human U6 promoter expressing oPL tg6 shRNA), hEF-1 tg2 (human elongation factor-1α promoter expressing oPL tg 2 shRNAmiR) and oPGK tg6 (ovine phosphoglycerate kinase-1 promoter expressing oPL tg 6 shRNAmiR), were selected to be tested in vivo. Day 9 blastocysts were harvested from naturally mated donor ewes, infected with one of the lentiviral constructs, and 2 to 3 blastocysts were surgically transferred to recipient ewes. At 55 dGA, uterine vein (UtV) blood and placental tissues were collected for analysis of oPL expression, and compared to naturally mated controls (NMC). Based on a 95% confidence interval created from UtV oPL concentrations in NMC pregnancies (n=4), 3 out of 4 hLL3.7 tg6 pregnancies, 3 out of 4 hEF tg2 pregnancies and 4 out of 4 oPGK tg6 pregnancies were classified as responder pregnancies. Compared to NMC pregnancies, UtV oPL concentrations were significantly reduced (P≤0.05) in responder pregnancies. While we hypothesized that oPL deficiency will result in IUGR near-term, at 55 dGA there were no differences in fetal weights. To test our overall hypothesis, we generated 8 hEF-1-SC (human elongation factor-1α promoter expressing scrambled control shRNAmiR), 9 hEF-1 tg2, 7 hEF-1 tg6 (human elongation factor-1α promoter expressing oPL tg6 shRNAmiR) and 9 hLL3.7 tg6 singleton pregnancies that were harvested at 135 dGA. Based on two standard deviations below the mean placental weight of the hEF-1 SC pregnancies, 2 out of 7 hEF-1 tg6 pregnancies and 6 out of 9 hLL3.7 tg6 pregnancies were classified as tg6 responder pregnancies (tg6). Tg6 pregnancies resulted in significantly decreased (P≤0.05) oPL mRNA concentrations, placental weight and fetal body weight compared to the controls at 135 days of gestation. These data confirm the effect of lentiviral-oPL targeting vectors and suggest that oPL plays a significant role in early placental development. Interestingly, we also observed that tg6 pregnancies had significantly increased (P≤0.05) placental efficiency relative to controls, which may function as a coping mechanism to maintain pregnancy in the face of oPL deficiency. Uterine artery to uterine vein glucose gradients were also increased (P≤0.05) in tg6 pregnancies compared to controls, which may be indicative of increased glucose uptake by the placenta in order to maintain function. Further analysis revealed that circulating fetal insulin tended to be decreased in the umbilical artery (P≤0.10) of tg6 fetuses relative to control fetuses, thus supporting a role for oPL in altering fetal insulin production. Finally, mRNA concentrations of insulin-like growth factors (IGF) -I and -II, and insulin-like growth factor binding proteins (IGFBP) -2 and -3 were significantly reduced (P≤0.05) in fetal liver tissue of tg6 fetuses compared to the controls. While this could be an indirect result of IUGR, oPL may well induce the expression of fetal insulin-like growth factors during in utero development. Based on these results, our hypothesis that oPL deficiency during gestation would result in intrauterine growth restriction appears correct. Surprisingly, it appears that oPL may have its greatest impact during early pregnancy when the placenta is developing, however the presence of adequate oPL is likely to be important throughout gestation for healthy fetal growth.
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    The impact of placental SLC2A3 (GLUT-3) RNA interference on fetal growth and physiology at mid-gestation in sheep
    (Colorado State University. Libraries, 2022) Lynch, Cameron S., author; Anthony, Russell V., advisor; Tesfaye, Dawit, committee member; Engle, Terry, committee member
    Glucose is the primary energy substrate for fetal oxidative processes and growth. In order for glucose to be transported from maternal to fetal circulation in the ruminant placenta, it must be sequentially transported by SLC2A1 (GLUT-1) on the maternal-fetal syncytial layer, then by SLC2A3 (GLUT-3) on the apical trophoblast membrane, and again by SLC2A1 on the basolateral trophoblast membrane. SLC2A1 is the most abundant placental facilitative glucose transporter, and as such, is believed to be the primary glucose transporter in human and sheep placenta. However, SLC2A3 exhibits a five-fold greater affinity and transport capacity for glucose. As such, in addition to its location on the apical trophoblast membrane, any deficiency in SLC2A3 could impact trophoblast glucose uptake and placental transfer of glucose to the fetus, thus potentially altering placental development and setting the stage for fetal hypoglycemia and intrauterine growth restriction (IUGR). It was our objective to use placenta-specific RNA interference (RNAi) to diminish SLC2A3, and determine the impact at mid- gestation (75 dGA) in sheep. The resulting pregnancies underwent a terminal surgery at 75 dGA. SLC2A3 RNAi resulted in a 37% reduction (p ≤ 0.05) in placental SLC2A3 concentration. SLC2A3-deficiency resulted in decreased fetal growth as evident by reduced fetal weight (p ≤ 0.10), head circumference (p ≤ 0.05), femur length (p ≤ 0.05), and tibia length (p ≤ 0.05). While there were no significant reductions in maternal glucose or insulin concentrations, the SLC2A3 RNAi pregnancies had decreased umbilical vein (p ≤ 0.05) and umbilical artery (p ≤ 0.05) glucose concentrations, as well as reduced umbilical artery insulin (p ≤ 0.10). Additionally, apparent attempts at compensation for SLC2A3-deficiency, by increasing SLC2A1, CSH, and IGF-2, were unable to prevent fetal hypoglycemia and the impacts on fetal development. Placental SLC2A1 concentration were increased (p ≤ 0.10), however this increase in expression was unable to prevent fetal hypoglycemia. The significant increase in umbilical vein CSH concentrations (p ≤ 0.05) appeared to preserve fetal liver weight and circulating umbilical concentrations of IGF-1, both of which are commonly decreased in IUGR pregnancies. SLC2A3-deficiency also resulted in a significant increase in IGF-2 (p ≤ 0.05), IGF1R (p ≤ 0.05), and IGF2R (p ≤ 0.05) expression. This suggests an apparent attempt to increase placental growth via IGF-2 acting through IGF1R, while IGF2R, which primarily acts to sequester and degrade IGF-2, doesn't allow placental growth to be overstimulated. While it has been suggested that SLC2A3 is predominantly important in late gestation, our data indicate that SLC2A3 is important for normal fetal development and appears to be a rate limiting glucose transporter during the first-half of gestation. A deficiency in SLC2A3 impacts trophoblast glucose uptake and subsequently glucose transfer to the fetus, and appears to set the stage during early gestation for the development of IUGR.
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    The physiological ramifications of chorionic somatomammotropin RNA interference
    (Colorado State University. Libraries, 2021) Tanner, Amelia R., author; Anthony, Russell V., advisor; Rozance, Paul J., advisor; Engle, Terry E., committee member; Thomas, Milton G., committee member; Winger, Quinton A., committee member
    To view the abstract, please see the full text of the document.
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    The role of proline rich 15 in trophoblast cell development
    (Colorado State University. Libraries, 2012) Gates, Katherine C., author; Anthony, Russell V., advisor; Clay, Colin, committee member; Duval, Dawn L., committee member; Hansen, Thomas R., committee member
    Maintenance of pregnancy in eutherian mammals requires a sophisticated and tightly regulated program of gene expression in order to develop a fully functional placenta. This transient organ mediates nutrient and gas exchange between the mother and fetus while protecting the fetus from the maternal immune system. Deviations from the normal regulation of gene expression during early pregnancy can lead to early embryonic loss as well as dysfunctional placentation, which can cause significant maternal and fetal morbidity and mortality. Proline rich 15 (PRR15) is a low molecular weight nuclear protein expressed by the trophoblast during early gestation in several mammalian species, including humans, mice, cattle, sheep, and horses. Immunohistochemistry revealed localization of PRR15 to the trophectoderm and extraembryonic endoderm of day 15 sheep conceptuses. In humans, PRR15 is localized in the nuclei of both first and second trimester trophoblast cells. Additional research has shown increased PRR15 transcription in colorectal cancers with mutations in the adenomatous polyposis coli (Apc) protein, suggesting a link to the Wnt signaling pathway. PRR15 mRNA concentrations increase when trophoblast cells, both sheep (oTR) and human (ACH-3P), are cultured on Matrigel, a basement membrane matrix. The expression profile in the sheep conceptus during pregnancy revealed a rise in PRR15 mRNA concentrations during the period of conceptus elongation with a peak in expression at day 16 of gestation, followed by a decline to day 30 of gestation. This peak coincides with a halt in elongation of the conceptus, and the initial period of apposition to the uterine luminal epithelium. Lentiviral-mediated knockdown of PRR15 in ovine trophectoderm at the blastocyst stage led to demise of the embryo by day 15 of gestation. This provides compelling evidence that PRR15 is a critical factor during this precarious window of development when initial attachment and implantation begin. The first aim of this research was to determine the effect of PRR15 deficiency on trophoblast gene expression, as well as trophoblast proliferation and survival. The human first trimester trophoblast cell line, ACH-3P, was infected with control lentivirus (LL3.7) and lentivirus expressing a short hairpin (sh)RNA to target PRR15 mRNA for degradation, resulting in a 68% decrease in PRR15 mRNA (p<0.01). Microarray analysis of these cell lines revealed differential expression of genes related to cancer, focal adhesion, and p53 signaling. We selected 21 genes for validation of mRNA levels by quantitative real-time RT-PCR, 18 (86%) of which gave results consistent with the microarray analysis, with similar direction and magnitude fold changes. This included significant up-regulation of GDF15, a cytokine increased in pregnancies with preeclampsia. GDF15 mRNA concentrations were examined more extensively during early ovine gestation, which revealed that GDF15 was low during peak PRR15 expression, then increased significantly at day 30 when PRR15 was nearly undetectable. Proliferation, as measured by cell metabolic activity and bromodeoxyuridine (BrdU) uptake, decreased in the PRR15-deficient cells, which was consistent with a decrease observed in cell cycle-related genes CCND1 and CDK6, and an increase in CCNG2 and CDKN1A in the PRR15-deficient cells. TNFSF10, a tumor necrosis factor superfamily member known to induce apoptosis, and its receptor, TNFRSF10b, increased significantly in the PRR15-deficient cells, suggesting trophoblast cells may be more susceptible to apoptosis when depleted of PRR15. Assays for caspase activity and annexin V staining revealed an increased population of apoptotic cells when treated with shRNA to target PRR15. These results suggest that PRR15 is required for driving trophoblast proliferation and survival during early development of the placenta, functions that are critical to early embryonic survival and successful placentation. The second experimental aim was to examine regions of the PRR15 promoter that are necessary for regulating its expression in trophoblast cells and to identify the role of Wnt signaling in PRR15 transcription. The 5'-flanking sequences from -824, -640, -424, -326, and -284 bp to +7 bp relative to the annotated transcription start site were amplified by PCR and ligated into the pGL3-Basic plasmid. These vectors were co-transfected into the first trimester human trophoblast cell line, ACH-3P, HT29 (human colorectal carcinoma), oTR, and BHK-21 (hamster kidney fibroblast) cells with a RSV-β-galactosidase vector control. In ACH-3P cells, transactivation of the luciferase reporter was maximal following transfections with the -326 construct (15.4 ± 4.8-fold). Significant promoter activity was absent in the -284, -424, and -640 constructs, but regained with the -824 construct (14.8 ± 5.8-fold). These results suggest that cis-acting elements within the proximal promoter of the PRR15 gene are essential for expression in trophoblast cells, requiring the regions from -284 to -326 and -640 to -824. DNase I footprinting and electrophoretic mobility shift assays were performed to identify transcription factor binding sites within these regions. Due to the potential link to the Wnt signaling pathway, cells were treated with an inhibitor to GSK3β, the kinase responsible for phosphorylation and proteasomal degradation of β-catenin. Inhibition of GSK3β decreased PRR15 mRNA concentrations and decreased transactivation of the luciferase reporter in all proximal promoter reporter constructs; this effect was mediated through β-catenin activity in the proximal 284 bases of the PRR15 5'-flanking region. Furthermore, trophoblast cell proliferation decreased after treatment with the GSK3β inhibitor. Electrophoretic mobility shift assays on the region from -98 to -68 revealed differential binding of nuclear proteins derived from ACH-3P cells grown in the presence or absence of the GSK3β inhibitor. These results reveal that canonical Wnt signaling inhibits the transcription of PRR15, mediated in part through the -98 to -68 region of the 5'-flanking region, and decreases proliferation in trophoblast cells. This indicates that suppression of Wnt signaling may be crucial during early trophectoderm outgrowth in order to allow significant transcriptional activation of PRR15 and conceptus survival.