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California State University, Northridge the Effects Of CALIFORNIA STATE UNIVERSITY, NORTHRIDGE THE EFFECTS OF LECTINS IN SEA URCHIN LYTECHINUS PICTUS DURING GASTRULATION IN LOW CALCIUM SEA WATER A thesis submitted in partial fulfillment of the requirements For the degree of Master of Science in Biology By Siavash Nikkhou December 2013 The thesis of Siavash Nikkhou is approved by: ---------------------------------------------------- ----------------------------------------- Dr. Aida Metzenberg Date ---------------------------------------------------- ----------------------------------------- Dr. Stan Metzenberg Date ---------------------------------------------------- ----------------------------------------- Dr. Steven B. Oppenheimer, Chair Date California State University, Northridge ii Acknowledgements I would like to sincerely thank Dr. Steven B. Oppenheimer for believing in me and being the best mentor and an advisor a graduate can ask for and with his well rounded knowledge in the field assisted me throughout the research. I would like to thank the entire Biology faculty more specifically I would like to thank Dr Karels, Dr. Aida Metzenberg and Dr. Stan Metzenberg for their support and encouragement and answering every questions. I would like to thank my colleagues in Dr. Oppenheimer’s lab for helping me throughout the project. I would like to express gratitude towards my family for their never ending support and especially would like to thank my girlfriend and my best friend, Shadi Asadabadi for extensive support and patience she has showed in my journey throughout the past two years through thick and thin and sticking by my side to achieve this goal. iii Table of Contents Signature Page ii Acknowledgement Page iii List of Tables v List of Figures vi Abstract x Introduction 1 Materials and Methods 14 Table of lectins 23 Results 24 Discussion 78 References 84 iv List of Tables Table 1: Lectins and their Specific Carbohydrate 23 Table 2: Embryo count for morphologies exhibited in control and experimental concentrations for lectins tested 25 Table 3: Summary of effects in control and experimental concentrations in embryos 68 Table 4: Comparison of t-test for mean percentage of morphologies for control and various concentrations for each lectin using two- tailed test 71 Table 5: Major Outcomes of Lectins 76 v List of Figures Figure 1A Control Lytechinus pictus embryos 48 hours after fertilization. 28 Figure 1B Control Lytechinus pictus embryos 48 hours after fertilization. 28 Figure 1C Control Lytechinus pictus embryos 48 hours after fertilization. 29 Figure 2A Lytechinus pictus embryos treated with 0.1 mg/ml of Artocarpus integrifolia 30 Figure 2B Lytechinus pictus embryos treated with 0.1 mg/ml of Artocarpus integrifolia 30 Figure 2C Lytechinus pictus embryos treated with 0.1 mg/ml of Artocarpus integrifolia 31 Figure 3A Lytechinus pictus embryos treated with 0.01 mg/ml of Artocarpus integrifolia 32 Figure 3B Lytechinus pictus embryos treated with 0.01 mg/ml of Artocarpus integrifolia 32 Figure 3C Lytechinus pictus embryos treated with 0.01 mg/ml of Artocarpus integrifolia 33 Figure 4A Lytechinus pictus embryos treated with 0.001 mg/ml of Artocarpus integrifolia 34 Figure 4B Lytechinus pictus embryos treated with 0.001 mg/ml of Artocarpus integrifolia 34 Figure 4C Lytechinus pictus embryos treated with 0.001 mg/ml of Artocarpus integrifolia 35 Figure 5A Lytechinus pictus embryos treated with 0.0001 mg/ml of Artocarpus integrifolia 36 Figure 5B Lytechinus pictus embryos treated with 0.0001 mg/ml of Artocarpus integrifolia 36 Figure 5C Lytechinus pictus embryos vi treated with 0.0001 mg/ml of Artocarpus integrifolia 37 Figure 6A Lytechinus pictus embryos treated with 0.00001 mg/ml of Artocarpus integrifolia 38 Figure 6B Lytechinus pictus embryos treated with 0.00001 mg/ml of Artocarpus integrifolia 39 Figure 6C Lytechinus pictus embryos treated with 0.00001 mg/ml of Artocarpus integrifolia 39 Figure 7A Control Lytechinus pictus embryos 48 hours after fertilization. 41 Figure 7B Control Lytechinus pictus embryos 48 hours after fertilization. 42 Figure 7C Control Lytechinus pictus embryos 48 hours after fertilization. 42 Figure 8A Lytechinus pictus embryos treated with 0.1 mg/ml of Triticum vulgaris 43 Figure 8B Lytechinus pictus embryos treated with 0.1 mg/ml of Triticum vulgaris 44 Figure 8C Lytechinus pictus embryos treated with 0.1 mg/ml of Triticum vulgaris 44 Figure 9A Lytechinus pictus embryos treated with 0.01 mg/ml of Triticum vulgaris 45 Figure 9B Lytechinus pictus embryos treated with 0.01 mg/ml of Triticum vulgaris 46 Figure 9C Lytechinus pictus embryos treated with 0.01 mg/ml of Triticum vulgaris 46 Figure 10A Lytechinus pictus embryos treated with 0.001 mg/ml of Triticum vulgaris 47 Figure 10B Lytechinus pictus embryos treated with 0.001 mg/ml of Triticum vulgaris 48 Figure 10C Lytechinus pictus embryos treated with 0.001 mg/ml of Triticum vulgaris 48 vii Figure 11A Lytechinus pictus embryos treated with 0.0001 mg/ml of Triticum vulgaris 49 Figure 11B Lytechinus pictus embryos treated with 0.0001 mg/ml of Triticum vulgaris 50 Figure 11C Lytechinus pictus embryos treated with 0.0001 mg/ml of Triticum vulgaris 50 Figure 12A Lytechinus pictus embryos treated with 0.00001 mg/ml of Triticum vulgaris 51 Figure 12B Lytechinus pictus embryos treated with 0.00001 mg/ml of Triticum vulgaris 51 Figure 12C Lytechinus pictus embryos treated with 0.00001 mg/ml of Triticum vulgaris 52 Figure 13A Control Lytechinus pictus embryos 48 hours after fertilization. 55 Figure 13B Control Lytechinus pictus embryos 48 hours after fertilization. 55 Figure 13C Control Lytechinus pictus embryos 48 hours after fertilization. 56 Figure 14A Lytechinus pictus embryos treated with 0.1 mg/ml of Phaseolus vulgaris PHA-L 57 Figure 14B Lytechinus pictus embryos treated with 0.1 mg/ml of Phaseolus vulgaris PHA-L 57 Figure 14C Lytechinus pictus embryos treated with 0.1 mg/ml of Phaseolus vulgaris PHA-L 58 Figure 15A Lytechinus pictus embryos treated with 0.01 mg/ml of Phaseolus vulgaris PHA-L 59 Figure 15B Lytechinus pictus embryos treated with 0.01 mg/ml of Phaseolus vulgaris PHA-L 59 Figure 15C Lytechinus pictus embryos treated with 0.01 mg/ml of Phaseolus vulgaris PHA-L 60 Figure 16A Lytechinus pictus embryos viii treated with 0.001 mg/ml of Phaseolus vulgaris PHA-L 61 Figure 16B Lytechinus pictus embryos treated with 0.001 mg/ml of Phaseolus vulgaris PHA-L 62 Figure 16C Lytechinus pictus embryos treated with 0.001 mg/ml of Phaseolus vulgaris PHA-L 62 Figure 17A Lytechinus pictus embryos treated with 0.0001 mg/ml of Phaseolus vulgaris PHA-L 63 Figure 17B Lytechinus pictus embryos treated with 0.0001 mg/ml of Phaseolus vulgaris PHA-L 63 Figure 17C Lytechinus pictus embryos treated with 0.0001 mg/ml of Phaseolus vulgaris PHA-L 64 Figure 18A Lytechinus pictus embryos treated with 0.00001 mg/ml of Phaseolus vulgaris PHA-L 65 Figure 18B Lytechinus pictus embryos treated with 0.00001 mg/ml of Phaseolus vulgaris PHA-L 65 Figure 18C Lytechinus pictus embryos treated with 0.00001 mg/ml of Phaseolus vulgaris PHA-L 66 Figure 19 Percentage of embryos exhibiting different morphologies for treated and control embryos using lectin Artocarpus integrifolia 73 Figure 20 Percentage of embryos exhibiting different morphologies for for treated and control embryos using lectin Triticum vulgaris 74 Figure 21 Percentage of embryos exhibiting different morphologies for for treated and control embryos using lectin Phaseolus vulgaris (PHA-L) 75 ix ABSTRACT THE EFFECTS OF LECTINS IN SEA URCHIN LYTECHINUS PICTUS DURING GASTRULATION IN LOW CALCIUM SEA WATER By Siavash Nikkhou Master of Science in Biology In order to help learn about the possible role of carbohydrate – containing molecules in gastrulation in the model sea urchin embryo (Lytechinus pictus), 24 hr sea urchin embryos were incubated with 3 lectins (carbohydrate binding proteins) Triticum vulgaris (wheat germ agglutinin), Artocarpus integrifolia agglutinin and Phaseolus vulgaris PHA-L agglutinin at 0.1-0.00001 mg/ml. Triticum is a specific binder of N-acetyl-D-glucosamine- like residues, as is Phaseolus, while Artocarpus is a specific binder of D-galactose-like residues. The embryos were treated with and without these lectins at all 5 concentrations for an additional 24 hrs at 15 ⁰C in lower calcium artificial seawater (that speeds entry of molecules into the interior of the embryos) in 96 wells well flat bottom microplates. The wells were treated with 10% formaldehyde to fix the embryos at the 48 hr stage (late gastrula). All embryos in each well were scored as to their morphologies: complete archenteron, incomplete archenteron, non-invaginated, exogastrulated or dead. Thousands of embryos were scored. Means of percentages of each morphology for each concentration of each lectin was plotted with standard error bars and a t-test was used to determine if any differences in the experimentals vs. controls were statistically significant (P<0.05). The results indicated statistically significant concentration dependent effects of all 3 lectins on altering the morphologies of the embryos. These preliminary results suggest that N-acetyl –D- glucosamine and D-galactose groups maybe involved in archenteron elongation and organization. The microplate assay is an effective means of quantitatively determining the precise effects of reagents on sea urchin morphologies, an NIH designated model for higher organisms including humans. x INTRODUCTION Sea urchins are model organisms used to provide insight into how organisms function and develop. Sea urchins are simple organisms that are translucent during the larval stage, allowing for observation of cell division and reproduction. Sea urchins are used so widely as a model organism because they provide an excellent model for the study of cell differentiation, reproduction, development, and cell adhesions (Gustafson et al., 1963). Sea urchins have been used as a model system for over a century. Their popularity as a research subjects comes from their widespread availability, the ease with which they can be stored, and the large numbers of gametes that can be harvested in a very short period of time.
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