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Molecular Analysis of the Epiphyseal Growth Plate in Rachitic Broilers: Evidence for the Etilogy of the Condition

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Molecular Analysis of the Epiphyseal Growth Plate in Rachitic Broilers: Evidence for the Etilogy of the Condition MOLECULAR ANALYSIS OF THE EPIPHYSEAL GROWTH PLATE IN RACHITIC BROILERS: EVIDENCE FOR THE ETILOGY OF THE CONDITION MASTER’S THESIS Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Julianne Eileen Rutt, B.A. ***** The Ohio State University 2008 Master’s Examination Committee: Approved by: Dr. David Latshaw, Advisor Dr. Kichoon Lee __________________________ Dr. Pasha Lyvers-Peffer Dr. David Latshaw Animal Sciences Graduate Program 2 ABSTRACT There is a lack of data in the literature concerning calcium-deficient rickets, which requires recognition due to to the economic and welfare concerns of leg weakness in broilers, as well as being an ideal disease model to study the effect of calcium on chondrocyte maturation. Broilers were raised on an adequate and calcium-deficient diet, and the rickets condition was confirmed using visual assessment, histology, and blood plasma analysis. The expression of known chondrogenic genes as well as genes from a previous rickets-based microarray was analyzed using real-time PCR with control and deficient growth plate chondrocytes. Indian hedgehog (Ihh) was decreased in rickets, parathyroid- hormone receptor (PTHR-1) was increased in rickets, and parathyroid hormone related- peptide (PTHrP) showed no difference. The calcium-sensing receptor had a 20-fold increased expression in rickets. Three of the four bone morphogenic proteins (Bmp) analyzed (-2, -4, -6) and both Bmp receptors were expressed lower in rickets. Eukaryotic elongation factor 1-δ showed a trend of being decreased in rachitic plates, and annexin-V and fibrillin-I were decreased in rickets. ATP analysis of the growth plates using a luciferase assay revealed a trend of ATP being increased in the rickets growth plates. An attempt at developing an in vivo chondrocyte cell culture model was unsuccessful primarily due to lack of cell proliferation and contamination. ii3 ACKNOWLEDGEMENTS I would like to thank Dr. Latshaw, for all of his help and support throughout my masters thesis. I had a lot of fun working with you, and I hope we keep in touch. I would also like to thank Dr. Lee for his use of the laboratory and for his daily technical advice….although it was not always the best news, you were always right. Also, thank you to Dr. Peffer, who helped me a lot with statistics and figures. Thanks to Jonghun Shin, for his patience and laboratory help. Also, thanks to my Pea (Jenny Campbell)…. we met on day one of our masters degree, and found a friend for life. No one else understood what I was going through like you did, and I could not have gotten through without you. Thank you to my mom and grandma, who used their prayers to me through the tough times. Thank you to my dogs, Nova and Alaska, for showing me what really matters at the end of the day. A special thanks to my fiancé Kiel, who was there every step of the way, and both suffered and celebrated with me. ii4i VITA March 16, 1984……………………………………Born-Cleveland, Ohio 2006………………………………………………B.A. Biochemistry, College of Wooster 2006……………………………………………….Research Assistant, Aquatic Ecology Laboratory, The Ohio State University 2006-2008………………………………………...Graduate Research Associate, The Ohio State University FIELDS OF STUDY Major field: Animal Sciences Continuing field: Veterinary Medicine iv5 TABLE OF CONTENTS Abstract…………………………………………………………………………...ii Acknowledgements…………………………………………………………….…iii Vita………………………………………………………………………………..iv Table of Contents………………………………………………………………….v List of Figures……………………………………………………………………vii List of Tables………………………………………………………………..……iv Chapters: 1. Introduction: Background and Significance…………………………...1 1.1 Introduction to the growth plate…………………………………...1 1.2 Molecular aspects of chondrocyte differentiation…………………3 1.3 Introduction to calcium……………………………………………9 1.4 Calcium and chondrogenesis……………………………………...12 1.5 Leg weakness in broiler chickens…………………………………15 1.6 Leg deformities: rickets and tibial dyschondroplasia……………..17 1.7 Broiler diets and mineral balance…………………………………18 1.8 Microarray analysis: Gene expression patterns in rickets………....22 1.9 Energy and the growth plate……………………………………….25 1.10 Chondrocyte cell culture…………………………………….……26 1.11 Research Significance and Project Objectives…………………....30 2. Materials and Methods…………………………………………………31 2.1 Materials……………………………………………………………31 2.2 Animals and Diets…………………………………………………..31 2.3 Growth plate analysis…………………………………………….…33 2.4 Blood samples………………………………………………………33 2.5 Histology……………………………………………………………34 2.6 Real-time PCR…………………………………………………...…34 2.7 ATP assay………………………………………………………..…37 2.8 Chondrocyte cell culture……………………………………………38 2.9 Alizarin red staining……………………………………………..…39 2.10 Statistical analysis…………………………………………………39 v6 3. Results…………………………………………………….……………40 3.1 Growth plate observations and histology…………………………..40 3.2 Blood analysis………………………………………………………43 3.3 Quantitative real-time PCR………………………………………..44 3.4 ATP assay………………………………………………………….49 3.5 Cell culture…………………………………………………………50 4. Discussion 4.1 Validation of rickets and control growth plate phenotypes………...53 4.2 Quantitative real-time PCR………………………………………...56 4.3 Validation of microarray expression data…………………………..66 4.4 ATP assay…………………………………………………………..69 4.5 Cell culture………………………………………………………….71 4.6 Future work…………………………………………………………74 References…………………………………………………………………………..75 vi7 LIST OF FIGURES Figure Page 1.1. Histological features of a human growth plate, showing the various stages of chondrocyte differentiation……………………... 2 1.2. Coordinated feedback mechanism and localization of chondrogenic genes in the growth plate…………………….............. 8 1.3. Calcium stores and hormonal regulation of calcium and phosphate metabolism……………………...……………………... 10 1.4 Model for proposed role of CaR in growth plate chondrocyte differentiation……………………...…………………….. 14 1.5. Current industry usage levels of calcium and nonphytate phosphorus in broiler starter diets……………………...……………… 21 3.1 Relative numbers of broiler chickens with normal, TD, and rickets growth plates……………………...……………………...... 41 3.2 Effect of a control and calcium-deficient diet on growth plate histology in broiler chickens……………………...……………... 42 3.3. Concentration of calcium and the calcium: phosphorus ratio, in the plasma of broiler chickens fed control and calcium-deficient diets…… 43 3.4 Concentration of plasma bicarbonate in broilers fed control and calcium-deficient diets…………………………………………….. 44 3.5 Relative mRNA expression of three control genes known to be involved in chondrocyte differentiation in control and rickets growth plate samples of broiler chickens…………………. 45 3.6 Relative mRNA expression of various genes analyzed in this study in the proliferative and hypertrophic fractions in control and rickets growth plate samples of broiler chickens…………. 46 3.7 Relative mRNA expression of the genes involved in the growth plate feedback loop in control and rickets growth plate samples of 18-21-day old broiler chickens.…………………….... 47 vi8i 3.8 Relative mRNA expression of the various Bmps and BmpR genes in control and rickets growth plate sample 48 of broiler chickens.……………………...……………………............... 3.9 Relative mRNA expression of the genes detected in the microarray in control and rickets growth plate samples of 49 18-21-day old broiler chickens.……………………...………………… 3.10 Number of attached chondrocytes from control and rickets growth plates measured 24 hours after plating…………........................ 51 3.11 Relative mRNA expression of type X collagen in chondrocyte cell culture over one sampling period.…………………... 52 4.1 Proposed model for the involvement of the chondrogenic genes in the development of rickets……………………. 66 vii9i LIST OF TABLES Table Page 1.1 Proteins found in the various stages of differentiation in the growth plate…………………...…………………...……………. 4 1.2 Vitamin and mineral requirements of broilers…………………............ 19 1.3 Microarray genes of interest…………………...…………………......... 22 2.1 Composition (%) of the basal diets for control and calcium-deficient experiments…………………...…………………..... 32 2.2 Primers used in this study…………………...…………………............. 36 3.1 ATP assay sample and mean values…………………...……………… 50 4.1 Summary of real-time PCR results: Gene expression in growth plates from broilers fed adequate and 57 calcium-deficient diets. …………………...…………………............... i10v CHAPTER 1 BACKGROUND AND SIGNIFICANCE 1.1 Introduction to the growth plate Bone growth occurs at growth plates, regions of specialized cartilage at the ends of long bones. The growth plate is positioned between the epiphyseal bone crest and metaphyseal boundary, and is the region responsible for the formation of cartilage cells and subsequent bone formation. Growth plate cartilage is the determining factor in the bone length and the rate of longitudinal bone growth. The process by which this cartilage is converted into growing bone is known as endochondral ossification, and begins during fetal development. Bone formation is initiated with prechondrogenic condensation, in which mesenchymal cells aggregate and form clusters of cells. These aggregates differentiate and become chondrocytes, the primary cell type of cartilage. Endochondral ossification is driven by the proliferation and differentiation of growth plate chondrocytes. This growth process can be divided into three phases: chondrogenesis, calcification, and osteogenesis. During chondrogenesis, cartilage cells are formed and mature in the growth plate. Chondrocytes
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