Determinants of Testicular Echotexture in the Sexually Immature Ram Lamb by Jennifer Lynn Giffin A Thesis presented to The Faculty of Graduate Studies of The University of Guelph In partial fulfilment of requirements for the degree of Doctor of Philosophy in Biomedical Sciences Guelph, Ontario, Canada © Jennifer Lynn Giffin, November, 2014 ABSTRACT DETERMINANTS OF TESTICULAR ECHOTEXTURE IN THE SEXUALLY IMMATURE RAM LAMB Jennifer Lynn Giffin Advisor: University of Guelph, 2014 Dr. P. M. Bartlewski Throughout sexual maturation, dynamic changes in testicular macro- and microstructure and reproductive hormone levels occur. Future adult reproductive capability is critically dependent on these changes; therefore, regular monitoring of pubertal testicular development is desirable. However, conventional methods of assessment do not permit the frequent and non- invasive examination of testicular function. Recently, scrotal ultrasonography in conjunction with computer-assisted image analysis has emerged as a potential non-invasive alternative for male reproductive assessment. In this procedure, testicular echotexture, or the appearance of the ultrasonogram, is objectively quantified on the basis of brightness or intensity of the minute picture elements, or pixels, comprising the image. In general, testicular pixel intensity increases with age throughout sexual maturation; however, periodic fluctuations occur. Changes in testicular echotexture are related to microstructural attributes of the testes and reproductive hormone secretion, but reports on these relationships have been inconsistent. Therefore, the overall objective of the studies presented in this thesis was to investigate how testicular echotexture and its associations with testicular histomorphology and endocrine profiles may be influenced by various factors including: i) scrotal/testicular integument; ii) blood flow/content; iii) stage of development; and iv) altered spermatogenic onset. Serial scrotal ultrasonograms, testicular biopsies or castrations and blood samples were collected from early postnatal or peripubescent ram lambs and analyzed to obtain echotextural, histomorphological and endocrine data, respectively. Testicular echotextural attributes were influenced by the presence of the scrotal and testicular integument, the stage of spermatogenic development (mitotic vs. post-mitotic) and the amount of germ cell heterogeneity present with or without retinoic acid exposure, but not by blood flow or content. Tubular and luminal size of the seminiferous tubules could be predicted from testicular pixel intensity using a simple linear regression model and demonstrated a moderate strength correlation with testicular echotextural parameters, as did a number of cellular and sub-cellular histomorphological attributes and reproductive hormone levels. These results clearly demonstrate the ability of testicular echotexture to detect experimentally and clinically relevant markers of testicular function. Computerized image analysis of scrotal ultrasonograms holds promise as a non-invasive diagnostic and prognostic tool and novel monitoring device of testicular histophysiology. ACKNOWLEDGEMENTS My heartfelt appreciation and gratitude go to Dr. Pawel Bartlewski for his guidance and support throughout my graduate studies and research involvement in the Department of Biomedical Sciences. I could not have had a more patient, kind and understanding advisor than you. Thank you for encouraging and inspiring me to reach for my fullest potential. It has been an honour and a delight working with you. Most sincere appreciation is also expressed to Dr. Ann Hahnel for sharing her vast knowledge and expertise, and for her unending interest and support in all my endeavors. The generous investment of time and effort by my advisory committee members, Dr. Neil MacLusky, Dr. Jim Petrik and Dr. Tracey Chenier, as well as my examination committee, is gratefully acknowledged. Your contributions have been essential to the successful completion of my degree and are greatly valued by me. My graduate study experience would not have been the same without the hard work, dedication and willingness to help of the faculty, staff and students in the Department of Biomedical Sciences and the wider University of Guelph community. In particular, I would like to thank Pam Hasson and Jeff McFarlane from the Ponsonby Sheep Research Station for attending to the needs of the experimental animals, Kevin Hogg for his technical assistance capturing ultrasound images, Helen Coates for her skilled help in performing histological and histochemical techniques, Kanwal Minhas for her laboratory assistance in processing tissue samples and previous members of the Bartlewski and Hahnel labs: Sabrina Sangupta, Stephanie Wilson, Sean Kamani, Karan Dhir, Vicki Watts, Andrew Bertolini, Bret McLeod, Kamila Skalski, Taylor VanDuzer and Longfei Gao for their participation in various research activities. Thanks also to those who were not directly involved with my research, but nonetheless contributed to the supportive learning environment I encountered. Financial support for the completion of this degree was generously provided by the Ontario Ministry of Agriculture, Food and Rural Affairs - University of Guelph Highly Qualified Personnel (OMAFRA-U of G HQP) Graduate Scholarship Program and funding of studies by OMAFRA and the Natural Sciences and Engineering Research Council of Canada is gratefully acknowledged. I have truly been touched by the love and support that my family has given to me over the years. Thanks especially to my parents, Don and Jean, for always believing in me and encouraging me to aspire to my dreams. Finally, I would like to thank God who has wonderfully orchestrated all the details and events encompassing the fulfillment of this degree and has enabled me to achieve far more than I could have ever imagined. iii TABLE OF CONTENTS ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................... iii TABLE OF CONTENTS ............................................................................................................... iv DECLARATION OF THE WORK PERFORMED .................................................................... viii LIST OF TABLES ......................................................................................................................... ix LIST OF FIGURES ........................................................................................................................ x LIST OF ABBREVIATIONS ....................................................................................................... xii 1. INTRODUCTION ...................................................................................................................... 1 1.1 Testicular Anatomy and Physiology in the Sexually Mature Ram .................................. 2 1.1.1 Testicular Macrostructure ......................................................................................... 3 1.1.2 Testicular Microstructure .......................................................................................... 6 1.1.3 Spermatogenesis ....................................................................................................... 8 1.1.4 Steroidogenesis ....................................................................................................... 13 1.1.5 Endocrine Regulation.............................................................................................. 14 1.2 Stages of Testicular Development in the Sexually Immature Ram Lamb ..................... 16 1.2.1 Prenatal Testicular Development ............................................................................ 16 1.2.2 Early Postnatal Testicular Development ................................................................. 18 1.2.3 Prepubertal Testicular Development....................................................................... 20 1.2.4 Postpubertal Testicular Development ..................................................................... 24 1.3 Scrotal Ultrasonography ................................................................................................. 25 1.3.1 Image Formation ..................................................................................................... 25 1.3.2 Image Analysis........................................................................................................ 28 1.3.3 Testicular Echotextural Trends ............................................................................... 30 iv 2. RATIONALE, OBJECTIVES AND HYPOTHESES .............................................................. 32 3. A STUDY OF INTEGUMENTARY, HISTOMORPHOLOGICAL AND HAEMODYNAMIC DETERMINANTS OF TESTICULAR ECHOTEXTURE IN RAM LAMBS ............................ 35 3.1 Abstract .............................................................................................................................. 36 3.2 Introduction ........................................................................................................................ 37 3.3 Materials and Methods ....................................................................................................... 39 3.3.1 Animals and Experimental Design ................................................................................. 39