Cellular and molecular aspects of the response of the testis to nutrition in sexually mature sheep Yongjuan Guan 21004548 This thesis is presented for the degree of Doctor of Philosophy of The University of Western Australia School of Animal Biology Institute of Agriculture March 2015 Declaration Declaration The work presented in this thesis is original work of the author, and none of the material in this thesis has been submitted either in full, or part, for a degree at this university or any other universities or institutions before. The experimental designs and manuscript preparation were carried out by myself after discussion with my supervisors Prof Graeme Martin, A/Prof Irek Malecki and Dr Penny Hawken. Yongjuan Guan March 2015 1 Contents Contents Summary ....................................................................................................................................... 4 Acknowledgements ....................................................................................................................... 8 Publications ................................................................................................................................. 10 Chapter 1: General Introduction ................................................................................................. 12 Chapter 2: Literature Review ...................................................................................................... 16 2.1 Male reproduction ............................................................................................................. 18 2.1.1 Organization of the testis ........................................................................................... 18 2.1.2 Spermatogenesis ........................................................................................................ 21 2.1.3 Physiological control of male reproduction .............................................................. 23 2.1.4 Morphological changes associated with puberty ....................................................... 25 2.2 Environmental factors affecting male reproduction .......................................................... 26 2.2.1 Photoperiod ................................................................................................................ 26 2.2.2 Social-sexual signals .................................................................................................. 28 2.2.3 Stress and temperament ............................................................................................. 29 2.2.4 Nutrition ..................................................................................................................... 30 2.3 Small RNAs affect spermatogenesis and germ cell apoptosis in testis ............................. 35 2.3.1 Small RNA categories ................................................................................................ 35 2.3.2 miRNAs and spermatogenesis .................................................................................... 37 2.3.3 miRNAs and germ cell apoptosis ............................................................................... 37 2.3.4 piRNAs affect spermatogenesis .................................................................................. 38 2.4 Alternative pre-mRNAs splicing affects spermatogenesis and apoptosis ......................... 38 2.4.1 Alternative pre-mRNA splicing and spermatogenesis ................................................ 39 2.4.2 Alternative pre-mRNA splicing and spermatogenesis ................................................ 40 2.5 Conclusions and hypotheses .............................................................................................. 41 Chapter 3: General Materials and Methods ................................................................................ 42 3.1 Experimental location ....................................................................................................... 42 3.2 Experimental animals ........................................................................................................ 42 3.3 Nutrition treatment ............................................................................................................ 42 3.4 Body mass and scrotal circumference ............................................................................... 43 3.5 Semen collection and processing ...................................................................................... 44 3.6 Semen analysis .................................................................................................................. 44 3.7 Tissue collection and preservation .................................................................................... 46 2 Contents 3.8 Sperm concentration in testicular tissue ............................................................................ 46 3.9 Morphometric and histological analysis ........................................................................... 46 3.10 Molecular analysis .......................................................................................................... 50 3.11 Bioinformatics analysis ................................................................................................... 52 Chapter 4: Under nutrition reduces spermatogenic efficiency and sperm velocity, and increases sperm DNA damage in sexually mature male sheep .................................................................. 57 4.1 Abstract ............................................................................................................................. 57 4.2 Introduction ....................................................................................................................... 58 4.3 Materials and methods ...................................................................................................... 60 4.4 Results ............................................................................................................................... 64 4.5 Discussion ......................................................................................................................... 71 Chapter 5: Under-nutrition decreases Sertoli cell function in sexually mature male sheep ....... 77 5.1 Abstract ............................................................................................................................. 77 5.2 Introduction ....................................................................................................................... 78 5.3 Materials and methods ...................................................................................................... 81 5.4 Results ............................................................................................................................... 90 5.5 Discussion ......................................................................................................................... 99 Chapter 6: Roles of small RNAs in the effects of nutrition on apoptosis and spermatogenesis in the adult testis ........................................................................................................................... 104 6.1 Abstract ........................................................................................................................... 104 6.2 Introduction ..................................................................................................................... 105 6.3 Materials and methods .................................................................................................... 107 6.4 Results ............................................................................................................................. 115 6.5 Discussion ....................................................................................................................... 125 Chapter 7: Functional changes in mRNA expression and alternative pre-mRNA splicing associated with the effects of nutrition on apoptosis and spermatogenesis in the adult testis .. 131 7.1 Abstract ........................................................................................................................... 131 7.2 Introduction ..................................................................................................................... 132 7.3 Materials and methods .................................................................................................... 134 7.4 Results ............................................................................................................................. 139 7.5 Discussion ....................................................................................................................... 148 Chapter 8 ................................................................................................................................... 155 General Discussion ................................................................................................................... 155 References ................................................................................................................................. 161 3 Summary Summary The reproductive system of small ruminants is affected by a variety of environmental factors, including socio-sexual signals, photoperiod and nutrition. Genotype controls the final outcome, but nutrition is particularly important in geographical regions where the quality and quantity of pasture are poor during