Browsing by Author "Anthony, Russell V., committee member"

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Investigation into the mechanisms of bone loss in a sheep model of osteoporosis
(Colorado State University. Libraries, 2024) Bisazza, Katherine T., author; Easley, Jeremiah T., advisor; Anthony, Russell V., committee member; McGilvray, Kirk, committee member; Goodrich, Laurie R., committee member; Nelson, Bradley B., committee member
Osteoporosis is the most common metabolic bone disease in humans and the leading cause of fragility fractures in the aging population. Given the invasiveness of researching bone diseases in people, appropriate animal models are essential to both build our understanding of the disease as well as examine novel therapeutics. While small animals and rodents are more commonly used as models in bone research, large animal models offer the ability to perform robust, long-term studies on bone quality with higher translational impact. Ovariectomized sheep are a well-established large animal model for osteoporosis because of the comparable bone size and microarchitecture that is shared with humans. While the ovariectomized sheep has been utilized for decades as a model for the study of bone, many gaps in the model characterization remain. Based on results from a preliminary literature search, we developed study objectives and hypotheses to expand upon current knowledge gaps in the characterization of the sheep model of osteoporosis. In order to test these aims, we performed a single 12-month in vivo study utilizing sixteen ewes. Osteoporosis was induced experimentally in ten of these ewes via ovariectomy followed by a 24-week regimen of high dose corticosteroids, while six ewes were used as healthy seasonal controls. In our first aim, we compared the bone density and microarchitectural changes in the osteoporotic animals as compared to healthy controls over the course of a year. Dual-energy x-ray absorptiometry (DXA) scans revealed significant bone density loss in the osteoporotic animals in both the lumbar spine and tibia as compared to control animals. We also noted significant microarchitectural changes in iliac crest bone biopsies of osteoporotic animals as indicated by micro-computed tomography (microCT), including decreased bone volume fraction, trabecular thickness, and trabecular number, as well as increased trabecular spacing. Additionally, we compared the use of quantitative computed tomography (QCT) and DXA to measure bone mineral density and correlated those findings with microarchitectural parameters in the osteoporotic animals. We demonstrated superior QCT sensitivity and specificity to subtle bone changes in the lumbar spine as compared to DXA, as well as demonstrated a higher correlation of QCT with iliac crest biopsy microarchitectural changes. The second aim of our study was to explore the systemic and clinical impacts of osteoporosis model development in our ten sheep compared to the healthy control animals. To test this aim, we collected blood, bone marrow, and body weights throughout the course of the year-long study. Osteoporotic animals demonstrated significant impacts to hematology and serology blood levels over the course of model development, primarily at 3 and 6-months when corticosteroids were at peak use. In particular, we note significant reductions in monocytes, lymphocytes, and eosinophils at 3-months with accompanying neutrophilia, as well as an increase in platelet count and volume. We also observed an increase in serum phosphorus and electrolytes, decrease in kidney enzymes and total protein, and an increase in select liver enzymes at 3 and 6-months in the same animals. Serum cortisol and estradiol were significantly depleted at 3 and 4-months, respectively, in the osteoporotic animals. However, estradiol levels were maintained to control levels for the remainder of the study. All these changes indicate disruptions to multiple physiologic systems over the course of osteoporosis induction in sheep which may highlight the acute effects of administering high-dose glucocorticoids. In the third and final aim of this study, we investigated the morphometrical and proteomic changes in the bone of sheep following osteoporosis induction over the course of a year. Histomorphometry of iliac crest bone biopsies revealed decreases in trabecular bone area in osteoporotic model animals compared to healthy controls, while negligible differences were observed in cortical bone morphometry. Initial global untargeted proteomic outputs identified a total of 4,765 proteins from the iliac bone biopsy samples, 909 of which were determined to be differentially expressed over the course of model development in our osteoporotic sheep. Pathway analysis of differentially expressed proteins (DEPs) revealed unique enriched pathways at all time points. Enrichment of biological processes such as monocyte differentiation, metabolic processes, regulation of chromosome condensation, and immune responses were noted throughout osteoporosis development. When comparing the 909 DEPs between time points, we identified seven downregulated proteins shared between all time points as compared to baseline in the osteoporotic animals (CTR9, INPP5D, CDK6, PPP2R5C, NUP133, ITPRIPL1, W5PH60_SHEEP). Pathway analysis of these shared proteins revealed enrichment of p53 signaling, mRNA surveillance, sphingolipid signaling, and P13K-Akt signaling pathways. This study was the first to report on the proteomic changes of bone in conjunction with morphometry assessments in a sheep model of osteoporosis. All three of our described experiments allowed us to successfully fill in some of the knowledge gaps in the characterization of a large animal model of osteoporosis by further assessing both macro and micro changes in ovariectomized and steroid-dosed sheep over the course of a year. Large animal preclinical models offer researchers the ability to compare bone changes in the same animals over time, allowing for a more comprehensive insight into the progression of postmenopausal and age-related bone loss. Understanding the mechanisms driving bone loss and systemic changes in osteoporosis disease progression could aid in future cellular therapy research and investigation of novel pathway targets for osteoporosis treatment in humans.
Open Access
Luteinizing hormone induced oocyte maturation initiates mRNA decay in cattle
(Colorado State University. Libraries, 2010) Walker, David Joshua, author; Seidel, George E., Jr., advisor; Clay, Colin M., committee member; Bruemmer, Jason E., committee member; Anthony, Russell V., committee member
Oocyte maturation is a complex process consisting of signal transduction, ultrastructural changes, and mRNA transcription, translation, storage, and degradation. In vitro-matured oocytes initiate maturation in response to removal from an inhibitory follicular environment while in vivo-matured oocytes mature in response to the LH surge. Oocytes matured in vivo lead to more successful embryo production than those matured in vitro. This research concerned study of expression levels and action of selected transcripts involved in RNA processing that occur in in vivo oocyte maturation. The first experiment focused on the inability of GnRH to induce oocyte maturation in superstimulated cows during the luteal stage of the estrous cycle. Superstimulated cows were treated with PGF2a and GnRH to induce in vivo maturation or were treated with GnRH without PGF2a to induce an LH surge at 0, 3, 12, or 24 h before aspiration. While treatment with GnRH caused an increase in LH over no GnRH treatment, it was a smaller increase than that observed in cows treated with PGF2a before GnRH treatment (P<0.001) (No GnRH; 0.84 ng/mL, GnRH: 9.45 ng/mL, PGF2a: 93.86 ng/mL). Thus, increases in LH were sufficient to initiate epiregulin mRNA transcription in granulosa cells (P<0.06) with the greatest expression levels after 6h. However, germinal vesicle breakdown did not occur as reliably in cows with an intact corpus luteum 23 h after GnRH (treated with PGF2a 36 h prior to GnRH injection) (GV stage oocytes; Oh; 79%, 6h: 58%, 12h: 83%, 24h: 60%, vs. controls 6%)(P<0.001). In addition, cAMP levels remained stable in oocytes from cows treated with GnRH in the presence of progesterone regardless of post injection time, while control oocytes had a slightly elevated level of cAMP (Oh: 4.95, 6h: 3.98, 24h: 4.12, control: 7.41 fmol) (P>0.1). Phosphodiesterase 3A mRNA levels were unaffected by any treatment (P>0.10). These data suggest that although the stimulatory signaling of LH and epiregulin occur, cAMP levels are unaffected by GnRH treatment in the presence of progesterone. The second experiment evaluated mRNA concentrations in bovine oocytes of four transcripts involved in RNA regulation in mammalian cells; CUG-BP, PARK, eIF-4E, and PAP-1. In vivo- and in vitro-matured oocytes were collected 0, 3, or 6 hours after initiation of maturation via GnRH injection for in vivo-maturation and aspiration from follicles for in vitro-maturation. eIF-4E and PARN mRNA concentrations increased over time in both in vitro- and in vivo-matured bovine oocytes (P<0.05). In vivo-matured oocytes contained more eIF-4E mRNA molecules than in vitro-matured bovine oocytes (P<0.10). CUG-BP and PAP-1 concentrations remained stable over the first 6 h of maturation and were similar in the in vivo- and in vitro-matured oocytes. The final experiment concerned deadenylation patterns for the cyclin B1 3’ untranslated region (UTR) and GDF-9 3’UTR with a poly(A) tail of 60 adenosines. Bovine oocytes were injected with radiolabeled constructs after 0, 5, or 19 h of maturation and then cultured for 0, 1, or 3 h. Radiolabeled RNA was recovered from oocytes after culture and analyzed for changes in construct size, reflective of deadenylation or general degradation. Cyclin B1 underwent deadenylation before being degraded regardless of the stage of oocyte maturation. Analysis of gels showed an intermediate with 0 adenosines (AO) in the Cyclin B1 injected oocytes, while those injected with GDF-9 displayed no such intermediate. These findings indicate that injected GDF-9 transcript remains stable with a poly(A) tail of 60 adenosines or is simply degraded randomly in bovine oocytes matured for 0, 5, or 19 h. Deadenylation of Cyclin B1 mRNA begins immediately in bovine oocytes resulting in degradation of the Cyclin Bl.
Open Access
Regulation of trophoblast stem cell maintenance and differentiation by LIN28 and AP-2γ
(Colorado State University. Libraries, 2010) Fromme, Brittany A., author; Winger, Quinton A., advisor; Bouma, Gerrit J., advisor; Bailey, Susan M., committee member; Anthony, Russell V., committee member
The placenta is a unique organ essential for survival of the fetus in all eutherian mammals. Failure to develop a normal placenta in humans can lead to diseases, such as pre-eclampsia, with high morbidity and mortality for both the mother and the fetus. These diseases are thought to be caused by abnormal proliferation and differentiation of cells in the placenta. A mouse trophoblast stem (TS) cell culture system is a useful tool in studying TS cell proliferation and differentiation into trophoblast giant cells (TGCs). TS cells cultured in proliferative media (70% conditioned media, 30% TS media, FGF4, and heparin sulfate) will remain proliferative, and TS cells cultured under differentiation media (100% TS media) will differentiate into TGCs. LIN28 is a protein that regulates mRNAs and miRNAs, and is abundantly expressed in many undifferentiated tissues. AP- 2y has been shown to be essential for TS cell maintenance and TGC formation. AP-2y null mutants display embryonic lethality at E7.5 due to a severely disrupted extraembryonic portion of the embryo. In TS cells, AP-2y has been shown to bind to the promoter region of Lin28. This study investigates the hypothesis that Liti28 and Ap-2y are necessary regulators of trophoblast stem cell maintenance and differentiation into TGCs. This study shows the pluripotency genes, Lin28, Sox2, and NrObl, to be differentially expressed in proliferating TS cells and differentiated TGCs. MiRNAs can be used as markers for proliferation or differentiation. 28 significantly different miRNAs were detected between TS cells and TGCs, 18 up-regulated in TGCs and 9 downregulated in TGCs. Expression of the miR-290 family, initially thought to be ES cell specific, was detected in proliferating TS cells suggesting TS cells have similar miRNA mediated regulation of proliferation compared to ES cells. The Let-7 family of miRNAs was found to be up-regulated in differentiated TGCs. The Let-7 family has been shown to be regulated by LIN28, where LIN28 prevents accumulation of mature Let-7 miRNAs. In this study Lin28 was highly expressed in proliferating cells and the Let-7’s are upregulated in differentiated TGCs. Lin28 function in TS cells was assessed by knocking down Lin28 using shRNA lentiviral technology. Lin28 knockdown TS cells were used to observe results of knockdown. We obtained a 78% reduction of Lin28 mRNA, but found that loss of Lin28 in TS cells did not affect morphology, proliferation or differentiation. AP-2y null TS cells grown in culture fail to differentiate morphologically into TGCs. Lin28, Sox2, and NrObl show no difference in expression when grown in conditions to differentiate the cells, indicating a failure of AP-2y null TS cells to differentiate into TGCs. RO3306 is a compound used to block Cyclin-dependent Protein Kinase 1 and force endoreduplication, causing TS cells to differentiate into TGCs. AP-2y null TS cells cannot be forced to differentiate into TGCs, and instead undergo cell death, when cultured with RO3306. Additionally, AP-2y null TS cells express the pluripotency markers Oct4, Stella, and Nanog which only are expressed in ES cells and germ cells. MiRNA profiling of AP-2y null TS cells indicates that cells in proliferative conditions resemble wild type counterparts, but when proliferative conditions are removed we observe an increase in expression of the ES cell specific miR-302 cluster. While there was no effect of proliferation in wild type cells, loss of Lin28 in AP-2y null TS cells via lentiviral knockdown leads to a partial rescue of TGC formation. This suggests that Liii28 must be down-regulated in order for TGC formation, and that AP-2y regulates Lin28 in TS cells. Taken together these data suggest a role for Lin28 in mouse TS cell proliferation and differentiation, where Lin28 must become down regulated in order for differentiation into TGCs. AP-2y has been shown to bind to the Lin28 promoter in TS cells; this regulation enables TS cell differentiation into TGCs. This study also shows the necessity of AP-2y for TS cell differentiation into TGCs; loss of AP-2y leads to a more pluripotent state rather than allowing for differentiation. Loss of AP-2y leads to expression of pluripotency markers Oct4, Nanog, and Stella, and the ES cell specific miR-302 cluster, indicating an increase in pluripotency. We conclude that AP-2y and LIN28 are essential molecular regulators of TS cell proliferation and differentiation.
Open Access
Tcfap2c regulation of primordial germ cell development
(Colorado State University. Libraries, 2011) Guttormsen, Jillian Bosick, author; Winger, Quinton A., advisor; Bouma, Gerrit J., committee member; Anthony, Russell V., committee member; Garrity, Deborah, committee member
The development of germ cells during embryonic development is driven by a complex expression pattern of genes. The transcription factor Tcfap2c is expressed in germ cells throughout development from specification to adult sperm and oocytes. Tcfap2c expression is first seen in primordial germ cells around embryonic day (E)6.75 and has been classified as a germ cell specification gene. This study implicates Tcfap2c as a potential key factor in germ cells during specification, proliferation, migration and differentiation. In order to investigate the role of Tcfap2c in germ cells, we utilized the Cre/loxP conditional gene mutation strategy. Cre/loxP allows us to overcome the early embryonic lethality that arises from loss of Tcfap2c in traditional knock-out mice by creating Tcfap2c null mutation in specifically-targeted tissues. We created Tcfap2c mutant mice using the epiblast-specific Sox2-Cre model. Mutant ovaries from this model failed to express both germ cell specific markers and meiotic markers at E12.5. Immunohistochemistry at E18.5 failed to detect the germ cell specific marker NOBOX or the meiotic protein SYCP3, which confirmed that Sox2-Cre, Tcfap2c mutant mice lacked germ cells at late embryonic stages. However, Sox2-Cre, Tcfap2c mutant mice die prior to or at birth preventing us from studying adult gonads from these mice. To this end we used tamoxifen inducible ERTM-Cre mice to create Tcfap2c mutation in adult animals. We assessed ERTM-Cre, Tcfap2c mutant animals for fertility and gametogenesis; surprisingly, fertility, spermatogenesis and oogenesis were not affected in Tcfap2c mutant gonads. These results show that Tcfap2c is not necessary for adult maturation of gonocytes to produce mature sperm and oocytes. However, Sox2-Cre, Tcfap2c mutants lack germ cells indicating that Tcfap2c is necessary during fetal germ cell differentiation. The Sox2-Cre model was limited because Tcfap2c was deleted in the entire embryo and the mutants died at birth. Prdm1-Cre was used to produce a mouse where Tcfap2c is only deleted in germ cells beginning around specification. Prdm1-Cre, Tcfap2c mutants initially specified germ cell-like cells at E7.5; however, by E8.5 the germ cell numbers were decreased and they had not initiated migration towards the genital ridges. By E9.5 few if any germ cells were observed in Prdm1-Cre, Tcfap2c mutants. At E12.5 no germ cells were seen in Prdm1-Cre, Tcfap2c mutant XX or XY gonads. Adult ovaries and testes from Prdm1-Cre, Tcfap2c mutant mice were noticeably smaller than littermate controls and showed no oogenesis or spermatogenesis. The Prdm1-Cre model showed that mutation of Tcfap2c results in loss of germ cells in embryos by E9.5 suggesting that Tcfap2c plays a role during germ cell specification, proliferation and migration. We identified Tcfap2c as an important factor during early germ cell development; however, Tcfap2c expression is observed in germ cells well past specification. We believe that Tcfap2c is present in germ cells during during fetal gonad differentiation because it plays a role in regulating the gene expression pathways necessary for this event. We show that Tcfap2c is expressed in germ cells during the period of fetal gonad differentiation. Gene expression analysis of gonads from E11.5-13.5 reveals Tcfap2c as the most highly expressed member of the Tcfap2 family member. Tcfap2c is a member of a transcription factor family that regulates gene expression by binding consensus sequences within target gene promoters. TCFAP2 binding sites are present in promoter regions of germ cell specific genes Cadherin1 (Cdh1) and Kit oncogene (Kit), as well as in the promoter regions of genes involved in regulating pluripotency High mobility group AT-hook 2 (Hmga2), Nanog homeobox (Nanog) and Lin28. Using chromatin immunoprecipitation we demonstrate that TCFAP2C binds the promoter regions of Cdh1, Kit, Hmga2, Nanog and Lin28. The interaction between TCFAP2C and the promoter regions of Cdh1, Kit, Hmga2, Nanog and Lin28 indicates that Tcfap2c likely plays a functional role in the regulation of these genes. These genes are necessary for germ cell survival, migration and pluripotency. In conclusion, our results provide a new understanding of the role of Tcfap2c during different stages of germ cell development from specification to differentiation.