University of North Dakota UND Scholarly Commons Theses and Dissertations Theses, Dissertations, and Senior Projects January 2016 Hypothalamus And Pituitary Gland Development In The ommonC Snapping Turtle, Chelydra Serpentina, And Disruption With Atrazine Exposure Kathryn Lee Gruchalla Russart Follow this and additional works at: https://commons.und.edu/theses Recommended Citation Russart, Kathryn Lee Gruchalla, "Hypothalamus And Pituitary Gland Development In The ommonC Snapping Turtle, Chelydra Serpentina, And Disruption With Atrazine Exposure" (2016). Theses and Dissertations. 2069. https://commons.und.edu/theses/2069 This Dissertation is brought to you for free and open access by the Theses, Dissertations, and Senior Projects at UND Scholarly Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of UND Scholarly Commons. For more information, please contact [email protected]. HYPOTHALAMUS AND PITUITARY GLAND DEVELOPMENT IN THE COMMON SNAPPING TURTLE, CHELYDRA SERPENTINA, AND DISRUPTION WITH ATRAZINE EXPOSURE by Kathryn Lee Gruchalla Russart Bachelor of Science, Minnesota State University, Mankato, 2006 A Dissertation Submitted to the Graduate Faculty of the University of North Dakota in partial fulfillment of the requirements for the degree of Doctor of Philosophy Grand Forks, North Dakota August 2016 Copyright 2016 Kathryn Russart ii iii PERMISSION Title Hypothalamus and Pituitary Gland Development in the Common Snapping Turtle, Chelydra serpentina, and Disruption with Atrazine Exposure Department Biology Degree Doctor of Philosophy In presenting this dissertation in partial fulfillment of the requirements for a graduate degree from the University of North Dakota, I agree that the library of this University shall make it freely available for inspection. I further agree that permission for extensive copying for scholarly purposes may be granted by the professor who supervised my dissertation work or, in his absence, by the Chairperson of the department or the dean of the School of Graduate Studies. It is understood that any copying or publication or other use of this dissertation or part thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of North Dakota in my scholarly use which may be made of any material in my dissertation. Kathryn Lee Gruchalla Russart July 15, 2016 iv TABLE OF CONTENTS LIST OF FIGURES ......................................................................................................... viii LIST OF TABLES ............................................................................................................. xi ACKNOWLEDGMENTS ............................................................................................... xiv ABSTRACT ..................................................................................................................... xvi CHAPTER I. INTRODUCTION .......................................................................................1 II. SEXUALLY DIMORPHIC DEVELOPMENT OF THE HYPOTHALAMUS AND PITUITARY GLAND IN THE SNAPPING TURTLE, CHELYDRA SERPENTINA ...................................8 Abstract ............................................................................................8 Introduction ......................................................................................8 Methods..........................................................................................18 Egg Collection ...................................................................18 Morphological Analysis of Pituitary Gland Development ......................................................................18 Sex Differences in Gene Expression ..................................21 Results……………………………………………………………27 Pituitary Development in the Snapping Turtle…………………………………………..27 Sex Differences in Gene Expression ..................................34 Discussion ......................................................................................46 v III. HIGH-THROUGHPUT IDENTIFICATION OF DEVELOPMENTAL AND SEXUAL DIMORPHISM WITHIN THE HYPOTHALAMUS AND PITUITARY GLAND IN THE SNAPPING TURTLE………………………………...55 Abstract…………………………………………………………..55 Introduction………………………………………………………56 Methods…………………………………………………………..63 Animal and Tissue Collection……………………………63 RNA Preparation and Sequencing……………………….64 Sequence Assembly and Differentially Expressed Gene Detection……………………………….65 Sequence and Functional Annotation……………………66 Quantitative PCR Validation…………………………….66 Results……………………………………………………………67 Quantitative PCR Validation .............................................77 Discussion………………………………………………………..77 IV. ATRAZINE ALTERS EXPRESSION OF REPRODUCTIVE AND STRESS GENES IN THE DEVELOPING HYPOTHALAMUS OF THE COMMON SNAPPING TURTLE, CHELYDRA SERPENTINA……………………83 Abstract…………………………………………………………..83 Introduction………………………………………………………84 Methods…………………………………………………………..89 Egg Collection and Treatment…………………………...89 Tissue Collection and RNA Isolation…………………....91 Reverse Transcription and quantitative PCR .....................92 vi Statistical Analysis .............................................................94 Results .............................................................................................95 Gene Expression in Turtle Embryo....................................95 Gene Expression in Hatchling Turtles ...............................97 Gene expression in 6-Month Old Turtles.........................101 Discussion .....................................................................................105 V. EFFECTS OF ATRAZINE ON EXPRESSION OF REPRODUCTIVE AND STRESS GENES IN THE PITUITARY GLAND OF THE SNAPPING TURTLE, CHELYDRA SERPENTINA………………………………....114 Abstract .........................................................................................114 Introduction ...................................................................................114 Methods .........................................................................................118 Reverse Transcription, quantitative PCR, and Statistical Analysis ....................................................121 Results ..........................................................................................124 Gene Expression in Turtle Embryos ................................124 Gene Expression in Hatchling Turtles .............................127 Gene Expression in Six-month Old Hatchling Turtles .............................................................130 Discussion ....................................................................................133 VI. EPILOGUE……………………………………………………………..140 REFERENCES…………………………………...……………………………….……142 1 vii LIST OF FIGURES Figure Page 2.1 Embryonic and hatchling samples collected for morphological analysis of pituitary gland development in the snapping turtle.. ............................................28 2.2 Invagination of the oral ectoderm starts at late stage 6/stage 7.. ..............................29 2.3 Invagination of the oral ectoderm forms Rathke’s pouch, the primordial adenohypophysis. ....................................................................................................30 2.4 Cellular proliferation and folding of Rathke’s pouch. ..............................................31 2.5 Formation of the pituitary gland.. .............................................................................33 2.6 Two-way ANOVA shows that ethanol treatment decreased expression of ESR1 in the hypothalamus of females and males at late stage 20 of embryonic development. ...........................................................................................36 2.7 Two-way ANOVA shows that ethanol decreased expression of Kiss1 in the hypothalamus of one-week old hatchlings. ...............................................................38 2.8 Two-way ANOVA shows that ethanol treatment decreased expression of Kiss1R in the hypothalamus in one-week old hatchlings..........................................39 2.9 Two-way ANOVA shows a significant interaction between ethanol treatment and sex. .....................................................................................................41 2.10 Two-way ANOVA shows that ethanol treatment increased expression of FSHb in the pituitary gland of males and females at late stage 20 of embryonic development. ...........................................................................................43 3.1 Differentially expressed genes between incubation temperatures at stage 20 of development and 4 months after hatching.. ..........................................................69 3.2 Heat map of transcripts that are differentially expressed in the hypothalamus and pituitary gland of snapping turtles, including both sexually dimorphic and developmentally regulated transcripts................................................................70 viii 3.3 GO terms associated with biological processes for developmentally differentially expressed genes between stage 20 and 4 month hatchlings incubated at a male-producing temperature. ...........................................71 3.4 GO terms associated with biological processes for developmentally differentially expressed genes between stage 20 embryos and 4- month old hatchlings incubated at a female-producing temperature. .......................72 3.5 Networks of genes that are sexually dimorphic in hypothalami