MIAMI UNIVERSITY the Graduate School Certificate for Approving The
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MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Deepti L. Kumar Candidate for the Degree: Doctor of Philosophy Director (Dr. Paul F. James) Reader (Dr. David G. Pennock) Reader (Dr. Susan Hoffman) Reader (Dr. Phyllis Callahan) Graduate School Representative (Dr. Neil D. Danielson) ABSTRACT SODIUM ION TRANSPORTERS IN SPERM: EPIGENETIC REGULATION OF THE SPERM-SPECIFIC ALPHA4 Na,K-ATPASE AND ROLE OF THE EPITHELIAL SODIUM CHANNEL ALPHA IN SPERM PHYSIOLOGY by Deepti L. Kumar Ion transporters play an important role in maintaining normal sperm physiology including motility and the ability to fertilize the egg. Mutations or deletions in some transporters have been shown to affect sperm motility and fertility in rodents and humans. The study of sperm ion transporters is therefore of significance as it can help explain some of the causes of male infertility and can also serve to identify targets for male contraception. The focus of this dissertation is on sodium ion transporters in sperm physiology, in particular the sperm-specific alpha4 Na,K-ATPase and the epithelial sodium channel alpha (ENaC alpha). Little is known about the mechanisms that regulate expression of the alpha4 Na,K-ATPase. Therefore, the main goal of the first part of the dissertation is to understand the mechanism(s) involved in the regulation of expression of the alpha4 Na,K-ATPase gene which encodes a sperm-specific protein essential for maintaining normal sperm motility and fertility. A number of germ-cell specific genes are known to be regulated by DNA methylation, one of the most extensively studied epigenetic modifications. Hence we sought to determine whether DNA methylation is involved in regulating the expression of the alpha4 Na,K-ATPase. The main goal of the second part of the dissertation is to determine the specific role of ENaC alpha in sperm development, motility and fertility. ENaC alpha and delta are the only 2 subunits thought to be present in sperm. Recent studies suggest that ENaC delta is a psuedogene in mice and therefore the protein may not be produced. In addition, due to the use of non-specific inhibitors, studies regarding the role of ENaC in capacitation and motility are not clear and require further investigation. Therefore we sought to determine whether ENaC alpha plays a specific role in sperm motility and fertility using a conditional knockout experimental approach. SODIUM ION TRANSPORTERS IN SPERM: EPIGENETIC REGULATION OF THE SPERM-SPECIFIC ALPHA4 Na,K-ATPASE AND ROLE OF THE EPITHELIAL SODIUM CHANNEL ALPHA IN SPERM PHYSIOLOGY A DISSERTATION Submitted to the Faculty of Miami University in partial Fulfillment of the requirements For the degree of Doctor of Philosophy Department of Biology by Deepti Kumar Miami University Oxford, Ohio 2014 Dissertation Director: Dr. Paul F. James Table of Contents Title………………………………………………………………………………………………...i Table of contents…………………………………………………………………………………..ii List of Tables ……………………………………………………………………………………..v List of Figures…………………………………………………………………………………….vi Acknowledgements……………………………………………………………………………..viii CHAPTER 1. INTRODUCTION……………………………………………………………….1 CHAPTER 2. DNA methylation regulates the expression of the gene encoding the alpha4 Na,K-ATPase (Atp1a4) A. INTRODUCTION AND RATIONALE………………………………………................14 B. MATERIALS AND METHODS………………………………………………………...17 a) DNA methylation analysis using bisulfite sequencing…………………………..17 b) Culturing of GC-1spg cells and mouse ES cells…………………………………17 c) 5-AzaC treatment of cells and quantitative real time PCR ……………………...18 C. RESULTS………………………………………………………………………………..20 a) A single differentially methylated intragenic CpG island (Mα4-CGI) is present in Atp1a4…………………………………………………………………………………...20 b) CpGs in the putative Atp1a4 promoter (Mα4-Promoter) are not differentially methylated in mouse kidney and sperm……………………………………………………………..22 c) Upregulation of Atp1a4 expression and demethylation of the Mα4-CGI and the Mα4- Promoter following treatment of GC-1spg cells with 5-azaC…………………………..23 ii d) Loss of DNA methyltransferase (Dnmt) enzymes results in upregulation of Atp1a4 expression and demethylation of the Mα4-CGI and the Mα4-Promoter in mouse ES cells……………………………………………………………………………………...26 D. DISCUSSION……………………………………………………………………………30 E. CONCLUSION…………………………………………………………………………..34 CHAPTER 3. Methylation-dependent and methylation–independent mechanism of regulation of Atp1a4 involving the promoter and intragenic CGI A. INTRODUCTION AND RATIONALE…………………………………………………39 B. MATERIALS AND METHODS………………………………………………………...42 a) Generation of Mα4-CGI and Mα4-Promoter reporter constructs……………………42 b) In vitro methylation of reporter constructs…………………………………………..43 c) Transient transfections and dual luciferase reporter assays………………………….43 d) Transcription elongation analysis……………………………………………………43 C. RESULTS………………………………………………………………………………..45 a) The Mα4-Promoter is a methylation-dependent germ-cell specific promoter………………………………………………………………………….45 b) Mα4-CGI does not function as an alternative promoter in GC-1spg cells………………………………………………………………………………46 c) Methylation of the intragenic Mα4-CGI inhibits transcription elongation efficiency…………………………………………………………………………48 d) The Mα4-CGI functions as a silencer element in germ cells but not in somatic cells………………………………………………………………………………51 D. DISCUSSION……………………………………………………………………………56 E. CONCLUSION…………………………………………………………………………..68 iii CHAPTER 4. Role of the epithelial sodium channel alpha (ENaC α) subunit in sperm development, motility and fertility A. INTRODUCTION AND RATIONALE…………………………………………………74 B. MATERIALS AND METHODS ……………………………………………………......77 a) Generation of mice with sperm-specific loss of ENaC α………………………...77 b) Genotyping of the ENaC α knockout mice………………………………………77 c) Western Blot on ENaC α wild type and knockout sperm total protein..................78 d) Hematoxylin and Eosin (H&E) staining and immunofluorescence……………...79 e) Sperm motility assays……………………………………………………………80 f) Breedings to test in vivo fertilization ability of ENaC α KO males……………...81 C. RESULTS………………………………………………………………………………..82 a) Stra-8 Cre mediated deletion of the ENaC α floxed allele results in loss of the ENaC α expression in mouse sperm……………………………………………..82 b) ENaC α is dispensable for normal testis/sperm development……………………84 c) ENaC α is dispensable for normal sperm motility……………………………….86 d) ENaC α is dispensable for normal fertility………………………………………88 D. DISCUSSION……………………………………………………………………………90 E. CONCLUSION…………………………………………………………………………..94 CHAPTER 5. CONCLUSIONS AND FUTURE DIRECTIONS……………………………96 REFERENCES………………………………………………………………………………...102 iv LIST OF TABLES CHAPTER 2 Table 2.1. Bisulfite sequencing primer list……………………………………………………...19 Table 2.2. qPCR primer list……………………………………………………………………..19 Table 2.3. List of the potential transcription factor binding motifs in the Mα4-Promoter……...35 CHAPTER 3 Table 3.1. List of the potential transcription factor binding motifs in the Mα4-CGI…………...68 CHAPTER 4 Table 4.1. Primers for ENaC α and Stra-8 Cre genotyping……………………………………..78 Table 4.2. Reproductive performance of ENaC α KO males compared to ENaC α WT males...88 Table 4.3. Assessment of the genotype of the sperm that contributed to each fertilization event...............................................................................................................................................89 v LIST OF FIGURES CHAPTER 1. Figure 1.1. Na,K-ATPase…………………………………………………………………………2 Figure 1.2. Epigenetic mechanisms of gene silencing…………………………………………....3 Figure 1.3. Methylation of CpG dinucleotide - An epigenetic signal…………………………….4 Figure 1.4. Distribution of CpG’s in the genome………………………………………………...5 Figure 1.5. The epithelial sodium channel (ENaC) and membrane topology…………………….9 CHAPTER 2. Figure 2.1. Location of the intragenic CGI in the Atp1a4 locus…………………………….......21 Figure 2.2. Mα4-CGI is differentially methylated in mouse sperm and kidney………………...21 Figure 2.3. The CpGs in the Mα4-Promoter are not differentially methylated in mouse sperm and kidney………………………………………………………………………………………..23 Figure 2.4. 5-Aza2-Deoxycytidine (5-azaC) treatment but not trichostatin A (TSA) upregulates Atp1a4 expression and reduces DNA methylation in GC-1spg cells……………………………25 Figure 2.5. Loss of DNA methytransferase enzyme 1 (Dnmt1) results in upregulation of Atp1a4 expression and reduced DNA methylation in mouse ES cells…………………………………..28 Figure 2.6. Loss of de novo methytransferase enzymes (Dnmt3a and Dnmt3b) results in increased Atp1a4 expression and reduced DNA methylation in mouse ES cells………………..29 CHAPTER 3. Figure 3.1. The Mα4-Promoter is a tissue-specific promoter that displays methylation dependent activity…………………………………………………………………………………………... 46 Figure 3.2. Mα4-CGI does not function as an alternative promoter in GC-1spg cells or NIH3T3 cells………………………………………………………………………………………………48 Figure 3.3. Mα4-CGI methylation decreases transcription elongation efficiency of the Atp1a4 transcript in HEK 293 cells……………………………………………………………………....50 Figure 3.4. The Mα4-CGI functions as a silencer in GC-1spg cells but not in HEK 293 cells…53 Figure 3.5. Methylation of Mα4-CGI alters its silencer activity in GC-1spg cells……………...54 vi Figure 3.6. Methylation of the Mα4-CGI does not alter its silencer activity with respect to Mα4- Promoter in GC-1spg cells……………………………………………………………………….55 Figure 3.7. A model for the regulation of Atp1a4 expression by the Mα4-Promoter and Mα4- CGI……………………………………………………………………………….........................65