University of Nevada, Reno Characterization of the Genes Essential for Mouse Spermatogenesis A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Cellular and Molecular Pharmacology and Physiology by Qiuxia Wu Dr. Wei Yan/Dissertation Advisor May, 2012 THE GRADUATE SCHOOL We recommend that the dissertation prepared under our supervision by Qiuxia Wu entitled Characterization of the Genes Essential for Mouse Spermatogenesis be accepted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Dr. Wei Yan, Advisor Dr. Seungil Ro, Committee Member Dr. Grant W. Hennig, Committee Member Dr. Sean M. Ward, Committee Member Dr. Claus Tittiger, Graduate School Representative Marsha H. Read, Ph. D., Associate Dean, Graduate School May, 2012 i Abstract Spermatogenesis is a complex process that starts with the proliferation of differentiated spermatogonia through mitotic division. Primary spermatocytes produced from differentiated spermatogonia enter the prolonged prophase of meiosis during which DNA is exchanged by homologous recombination. Primary spermatocytes undergo two meiotic divisions to form haploid spermatids. Haploid spermatids differentiate through the elongation phase and eventually form mature spermatozoa through dramatic morphological changes termed spermiogenesis, during which: 1) the Golgi apparatus forms the acrosome, 2) nuclear chromatin undergoes compaction and condensation, 3) sperm tail is formed and 4) the excess cytoplasm of spermatid is eliminated. Given the complexity of spermatogenesis, the normal development of male germ cells is controlled by both protein-coding genes and small non-coding RNAs. Because of the lack of in vitro models, mouse models are currently the most powerful tools used to study these processes. KLHL10 (Kelch-like 10) is a spermatid-specific protein, which belongs to a BTB (Brica-brac, Tramtrack, and Broad-Complex)-Kelch protein superfamily. Haploinsufficiency of Klhl10 causes male infertility and prevents the genetic transmission of both mutant and wild-type alleles in mice. Therefore, a transgenic rescue strategy was used to overcome the haploinsufficiency of Klhl10 and knockout (KO) mice were generated for studying the function of Klhl10 during spermatogenesis. Klhl10 KO testes exhibited disrupted spermatogenesis, characterized by severe depletion of germ cells, degeneration of spermatids and reduction in the number of late spermatids. In ii Klhl10 KO testes, spermatid differentiation was arrested in elongating stage at step 9. Comparison of protein profiles between control and KO testes revealed that many mitochondrial proteins were up-regulated in Klhl10 KO testes. In addition, COX IV (mitochondrion marker) staining showed enhanced mitochondria signal in KLHL10 depleted germ cells. Our results indicate that KLHL10 might be involved in regulating mitochondrial protein turnover during late spermiogenesis. The BTB domain of KLHL10 was reported to directly interact with CUL3 (an ubiquitin E3 ligase), suggesting KLHL10 is involved in the ubiquitination pathway to regulate protein turnover. Using yeast two-hybrid, we screened an adult mouse testis library to identify testicular proteins that can interact with KLHL10 and spermatogenesis- associated proteins 3 (SPATA3) and 6 (SPATA6) were two proteins identified. In vitro co-immunoprecipitation assay revealed that KLHL10 interacts with both SPATA3 and SPATA6 through the Kelch domain, a substrate-recruiting domain in most of the CUL3- BTB/Kelch E3 ligase complexes. Therefore, an in vivo ubiquitination assay was performed to examine whether KLHL10 can recruit SPATA3 or SPATA6 for ubiquitination. We found the ubiquitination level of SPATA3, but not SPATA6, was significantly increased upon overexpression of KLHL10, suggesting SPATA3 is a substrate of CUL3-KLHL10 E3 ligase. Our data suggests CUL3-KLHL10 complex is a spermatid-specific ubiquitin E3 ligase that involved in removal of proteins during late spermiogenesis. Hils1 is another spermatid-specific gene, which encodes a linker histone H1-like protein. The expression of HILS1 overlaps with the expression of transition nuclear proteins (TNP1 and TNP2). While Hils1-/- males were fertile and Tnp1-/- males were iii subfertile, the double KO (Hils1-/-Tnp1-/-) mice we generated were completely infertile. Electron microscopy revealed severe nuclear condensation defect in both late spermatids and epididymal sperm of Hils1-/-Tnp1-/- mice. The number of epididymal sperm was highly reduced and most sperm had abnormal morphologies with head-bent-back as predominant defect in Hils1-/-Tnp1-/- mice. Double KO sperm also had a greater susceptibility of DNA to denaturation and elevated levels of protamine 2 precursors. Injection of mutant cauda epididymal sperm into intact oocyte showed normal fertilization rates, however, most zygotes didn’t develop beyond the 2-cell stage. Single cell PCR revealed that the mRNA expression profile was altered in 2PN and 2-cell mutant embryos compared to WT, suggesting the disruption of paternal nuclear condensation can cause abnormal embryo development at pre-implantation stage. In addition to gene functional study using universal KO mouse models, we also used conditional KO mouse models to study the functions of small RNAs during spermatogenesis. microRNAs (miRNAs) are produced from short hairpin structures by the cleavages of DROSHA, a RNase III enzyme, in the nucleus and DICER, another RNase III enzyme, in the cytoplasm. endo-siRNAs are distinguished from miRNAs in that endo-siRNAs are processed from naturally occurring long dsRNAs and the biogenesis of endo-siRNAs is DROSHA-independent but DICER-dependent. To investigate the role of miRNA and/or endo-siRNA in spermatogenesis, we generated Drosha or Dicer conditional knockout (cKO) mouse lines using Cre-loxP strategy to specifically delete Drosha or Dicer in spermatogenic cells in postnatal testes. Although both Drosha and Dicer cKO males are infertile, Drosha cKO testes appeared to display more severe spermatogenic disruptions than Dicer cKO testes. Microarray analyses iv revealed transcriptomic differences between Drosha and Dicer-null pachytene spermatocytes or round spermatids. Although levels of sex-linked mRNAs were mildly elevated, meiotic sex chromosome inactivation appeared to have occurred normally in both Drosha and Dicer cKO cells. Our data demonstrate that gene regulation mediated by small RNAs is required for the normal development of male germ cells and male fertility. Overall, we demonstrated that both protein-coding genes and small RNAs play roles in regulation of male germ cell development. Investigations using mouse models help us gain deeper understanding of the fundamentals of reproductive biology, which will ultimately benefit the human health. v Acknowledgements I would like to thank my dissertation advisor Dr. Wei Yan for accepting me to his research group and guiding me on my research. His instruction, support and encouragement make this dissertation possible. Sincere appreciation goes to my committee members Dr. Seungil Ro, Dr. Grant W. Hennig, Dr. Sean M. Ward and Dr. Claus Tittiger for their directions on my graduate progression and critical review of my qualifier proposal and this dissertation. I thank the previous and current members of Yan lab for their generous help and contributions to the project. I want to thank my friends in Reno for traveling with us, tasting gourmet food with us and having fun time with us. Special thanks goes to Shouhua Wang's family for their help and care. I would like to thank my parents Mr. Zhangxin Wu and Mrs. Xueqin Wu who were always supporting me and encouraging me with their best wishes. Finally I would like to thank my husband, Rui Song. He was always there supporting me and stood by me through good times and bad. vi Table of Contents Abstract………………………………………………………………………………….....i Acknowledgements………………………………………………………………………..v Table of Contents…………………………………………………………………………vi List of Tables……………………………………………………………………………..ix List of Figures…………………………………………………………………………......x List of Abbreviations……………………………………………………………………xiii Chapter 1: Introduction………………………………………………………………...….1 Human infertility and mouse spermatogenesis……………………………………1 Knockout strategies………………………………………………………………..3 Spermatogenesis studied by knockout mice: protein-coding genes………………5 Spermatogenesis studied by knockout mice: small non-coding RNAs…………...8 Translation to clinical practice and directions for the future……………………...9 Chapter 2: Lack of Klhl10, a spermatid-specific gene, causes male infertility………….20 Abstract…………………………………………………………………………..20 Keywords……………………………………………………………………...…21 Introduction………………………………………………………………………22 Materials and Methods………………………...………………………………....25 Results………...................………………………………...…………….……….28 Disccusion………...................…………………………………………………...33 References………...................…………………………………………………...45 Chanpter 3: KLHL10 targets SPATA3 for ubiquitination by a CUL3-based E3 ligase during mouse spermiogenesis………...................………………………………..............48 vii Abstract……...................………………………………………………………...48 Keywords………...................……………………………………………………49 Introduction………...................……………………………………………….....50 Materials and Methods………..........…………………………………………….53 Results………...................……………………………………………………….57 Discussion………...................…………………………………………………...61 References………...................…………………………………………………...68 Chapter 4: Genetic