The Pennsylvania State University The Graduate School Intercollege Graduate Degree Program in Physiology THE IMPACT OF ZINC DEFICIENCY DURING OOGENESIS, FOLLICLE ASSEMBLY, AND GROWTH A Dissertation in Integrative and Biomedical Physiology by James M. Hester 2018 James Hester Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2018 ii The dissertation of James Hester was reviewed and approved* by the following: Francisco J. Diaz Associate Professor of Reproductive Biology Dissertation Advisor Chair of Committee Wendy Hanna-Rose Associate Professor and Department Head Biochemistry and Molecular Biology Alan L. Johnson Walther H. Ott Professor in Avian Biology Claire M. Thomas Associate Professor of Biology and Biochemistry and Molecular Biology Donna H. Korzick Professor of Physiology and Kinesiology Chair, Intercollege Graduate Degree Program in Physiology *Signatures are on file in the Graduate School iii ABSTRACT The ovarian follicle is the fundamental unit of the ovary and of female reproduction in mammals. Each follicle contains one oocyte enclosed in somatic cells and follicle number is determined during fetal development in humans. Growth and development of ovarian follicles (folliculogenesis) is necessary to produce viable gametes as well as ovarian hormones including estrogen and progesterone. Folliculogenesis begins in the fetal ovary and may span several decades of life. Environmental and nutritional factors that affect folliculogenesis have the potential to impact health and fertility, and may be a source of new biotechnological innovation. One such factor, zinc, has previously been found to impact the final stages of follicle development including meiotic division, ovulation, epigenetic modification, fertilization, and embryo development. However, the role of zinc during the early stages of folliculogenesis have not been evaluated. To investigate the zinc requirement during early follicle development, we have employed 2 model species (C. elegans and Mus musculus) over the course of three separate studies of zinc deficiency. Study 1: We sought to introduce a new model organism to the study of micronutrient-mediated infertility. The roundworm C. elegans contains a unique germline in which oocytes are constantly generated in the adult, rather than only during fetal development as in mammals. To test the zinc requirement of this model, we pretreated nematode growth media with the zinc chelator TPEN. C. elegans grown on zinc deficient media showed a severe reduction in fertility (fewer progeny produced per hermaphrodite), defects in oocyte development, and disruption of the pachytene-to-diplotene meiotic transition. All effects were rescued by the addition of exogenous zinc to the TPEN treated media. This is the first time that a zinc requirement for early meiotic development has been shown in any model species. Study 2: To test the effect of zinc deficiency on growing follicles, we collected preantral ovarian follicles from 14-day old mice and cultured them for up to 10 days in control, or zinc deficient media. iv Zinc restriction altered follicle growth, particularly proliferation of somatic cells surrounding the oocyte. Zinc-deficient follicles also displayed higher rates of apoptosis in somatic cells, impaired paracrine signaling, and a reduced ability of the oocyte to undergo meiotic division – a prerequisite to fertilization. The detrimental effects of zinc chelation were rescued by the addition of exogenous zinc. Study 3: In mammals, the primordial follicle pool is established in the fetal ovary when mitotically derived germ cells irreversibly enter meiosis and become enclosed in somatic pregranulosa cells. Revealing a zinc requirement during this phase of development would be significant, as many pregnant women worldwide are marginally zinc deficient. To test the effects of zinc deficiency of primordial follicle formation, we collected fetal mouse ovaries on embryonic day 16.5 and cultured them in control, zinc deficient, or rescue media for 5 days to evaluate meiotic progression of the oocytes. We also collected newborn mouse ovaries and cultured them for 4 days under the same treatment conditions to evaluate the breakdown of germ cell nests into individual follicles. Zinc deficiency did not affect development in the fetal ovary. There were no changes in meiotic progression, oocyte survival, or gene expression. Zinc deficiency had a larger impact during newborn ovary culture. Zinc deficiency impaired germ cell nest breakdown leading to fewer primordial follicles. Gene expression and follicle activation were also affected by zinc deficiency. These effects were rescued by exogenous zinc. Taken together, these studies demonstrate a conserved zinc requirement for oocyte development beginning at earlier stages of oogenesis that previously observed. If these results translate to humans, impaired follicle development and impaired preantral follicle growth may contribute to infertility as a result of zinc deficiency. v TABLE OF CONTENTS LIST OF FIGURES ............................................................................................................ viii LIST OF TABLES .............................................................................................................. x ACKNOWLEDGEMENTS ................................................................................................ xi Chapter 1 Introduction ....................................................................................................... 1 Hypotheses .................................................................................................................. 2 Chapter 2 Literature Review............................................................................................... 3 Ovarian Follicle Development ..................................................................................... 4 Primordial Follicle Assembly ............................................................................... 6 Primordial Follicle Regulation ............................................................................. 11 Preantral Follicle Growth ..................................................................................... 12 Antral Follicle Growth ......................................................................................... 14 Oocyte-Granulosa Cell Communication ............................................................... 16 Ovulation ............................................................................................................. 18 Reproduction in Caenorhabditis. elegans..................................................................... 20 Zinc in Biology and Reproduction ............................................................................... 23 Zinc Biology ........................................................................................................ 23 Zinc in Reproduction ........................................................................................... 25 Human Zinc Deficiency – A Public Health Concern ............................................. 26 Conclusion .................................................................................................................. 27 Chapter 3 Zinc deficiency reduces fertility in C. elegans hermaphrodites and disrupts oogenesis and meiotic progression ..................................................................... 28 Abstract ....................................................................................................................... 29 Introduction ................................................................................................................. 30 Materials and Methods ................................................................................................ 32 C. elegans culture ................................................................................................ 32 Effect of TPEN exposure on progeny production .................................................. 32 Embryo number and oocyte development ............................................................. 34 DAPI staining ...................................................................................................... 34 Brightfield imaging for representative images ...................................................... 35 Statistics .............................................................................................................. 35 Results ........................................................................................................................ 35 TPEN exposure reduces progeny output in C. elegans .......................................... 35 Zinc restriction lowers the number of developing embryos in the uterus ............... 36 Zinc restriction disrupts oocyte development ........................................................ 38 Zinc restriction leads to an expanded region of pachytene germ cells in the proximal gonad ............................................................................................ 39 Discussion ................................................................................................................... 40 Acknowledgements ..................................................................................................... 43 vi Chapter 4 Zinc deficiency disrupts in vitro maturation of preantral ovarian follicles ........... 44 Abstract ......................................................................................................................