University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2018 Intergenerational Mechanisms Of Paternal Stress Transmission Jennifer C. Chan University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Neuroscience and Neurobiology Commons, and the Pharmacology Commons Recommended Citation Chan, Jennifer C., "Intergenerational Mechanisms Of Paternal Stress Transmission" (2018). Publicly Accessible Penn Dissertations. 2790. https://repository.upenn.edu/edissertations/2790 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/2790 For more information, please contact [email protected]. Intergenerational Mechanisms Of Paternal Stress Transmission Abstract Evidence that the intergenerational transmission of parental experiences can influence offspring outcomes prompts new consideration for the molecular mechanisms underlying disease risk and resilience. The role of the paternal preconception environment has been of particular interest, stimulating characterization of germ cell epigenetic marks that can respond dynamically to environmental insults and transmit this information at fertilization. Given such exciting potential for sperm epigenetic marks, how these marks are changed by the environment and subsequently impact offspring development are key questions that require investigation. In this dissertation, we address these questions using our established mouse model of paternal stress, where specific sperm microRNA altered by paternal chronic stress exposure causally reprogram offspring hypothalamic-pituitary-adrenal (HPA) stress axis reactivity and the hypothalamic transcriptome. First, we examined the role of glucocorticoids, a major component of the HPA stress response, as a signal for sperm microRNA changes. To ensure similar levels of glucocorticoids are produced in response to stress and thus are available for paternal cellular signaling, we developed an approach to confirm the stress sensitivity and reactivity of experimental mice. We next demonstrated that glucocorticoids are involved in communicating stress to the caput epididymis, a somatic tissue that secretes extracellular vesicles (EVs) to deliver microRNA from epididymal epithelial cells to maturing sperm. Using an in vitro model where we administered glucocorticoids to caput epididymal epithelial cells, we showed altered EV microRNA content and within epithelial cells, changes to histone post-translational modifications and increased glucocorticoid receptor levels, mimicking aspects of our in vivo paternal stress model. Further, we demonstrated the crucial role of caput epididymal glucocorticoid receptors in paternal stress transmission by transgenic knockdown, preventing offspring HPA axis and hypothalamic programming. In our final study, we provided evidence for the specificity of paternal stress sperm microRNA effects on embryonic brain and placental transcriptomes, indicating a tightly regulated process by which sperm microRNA are coordinated and function to influence offspring development. Together, the research presented in this dissertation provides insight into the mechanisms contributing to paternal transmission and support the paternal preconception environment as an influential factor in offspring disease risk and resilience. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Pharmacology First Advisor Tracy L. Bale Keywords Epigenetics, Extracellular vesicles, Intergenerational, Paternal, Stress Subject Categories Neuroscience and Neurobiology | Pharmacology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/2790 INTERGENERATIONAL MECHANISMS OF PATERNAL STRESS TRANSMISSION Jennifer C Chan A DISSERTATION in Pharmacology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2018 Supervisor of Dissertation _______________________ Tracy L. Bale, PhD Professor of Neuroscience Graduate Group Chairperson _______________________ Julie A. Blendy, PhD Professor of Pharmacology Dissertation Committee Zhaolan (Joe) Zhou, PhD, Associate Professor of Genetics R. Christopher Pierce, PhD, Professor of Neuroscience Rebecca A. Simmons, MD, Professor in Neonatology P. Jeremy Wang, PhD, Professor of Developmental Biology ACKNOWLEDGMENT I have been privileged to be in the company of great scientists, amazing friends, and the most loving family, without all of whom graduate school would have been much tougher and much less fun. While there have been numerous mentors and friends who have advised and supported me along the way, I must call out a few in particular. First and foremost, my mentor, Tracy Bale, shaped my scientific development and thinking, building my independence and confidence as a scientist. Her enthusiasm for our work, leading her once to pull over on a highway to email me about an idea, motivated me to push through the minute agitations of bench work. Most of all, she supported, and even encouraged, my ideas that may have been a little outside of the box, which made pushing the barriers of our research all the more enjoyable. My lab mates have been crucial to my success and sanity – Katie and Bridget, in particular, especially for the dozens of donuts you both have provided for the lab. Many of the conversations we had got me through the day, whether it was morning catch-ups with Katie or coming up with farfetched but awesome experiments with Bridget. I also need to acknowledge Eldin, for late scientific discussions and explaining Random Forests for the hundredth time to me; Chris H. for being my go-to helper and lunch buddy; and Jess for bearing with me as I scattered my things all over her desk. I am especially lucky to have met through the Pharmacology Graduate Group my incredible friends – Christie, Madhu, and Mia. These ladies treaded the highs and lows of the PhD life with me, and helped me enjoy/remember life outside of grad school. ii My family has been the most supportive and encouraging of this entire process. I am fortunate for my parents who, through their and my working long hours in our family restaurant, have emphasized to me the value of higher education and hard work, which got me through many long days. I am also thankful for my siblings, especially Priscilla, who has bore the burden of all my unleashed feelings and, despite that, has stayed by my side all this time. Lastly, this whole experience would have been much more difficult without my partner and soon-to-be husband, Phil, who routinely volunteers to listen to me practice for big science talks, during which he has only fallen asleep once, for now. iii ABSTRACT INTERGENERATIONAL MECHANISMS OF PATERNAL STRESS TRANSMISSION Jennifer C Chan Tracy L. Bale Evidence that the intergenerational transmission of parental experiences can influence offspring outcomes prompts new consideration for the molecular mechanisms underlying disease risk and resilience. The role of the paternal preconception environment has been of particular interest, stimulating characterization of germ cell epigenetic marks that can respond dynamically to environmental insults and transmit this information at fertilization. Given such exciting potential for sperm epigenetic marks, how these marks are changed by the environment and subsequently impact offspring development are key questions that require investigation. In this dissertation, we address these questions using our established mouse model of paternal stress, where specific sperm microRNA altered by paternal chronic stress exposure causally reprogram offspring hypothalamic-pituitary- adrenal (HPA) stress axis reactivity and the hypothalamic transcriptome. First, we examined the role of glucocorticoids, a major component of the HPA stress response, as a signal for sperm microRNA changes. To ensure similar levels of glucocorticoids are produced in response to stress and thus are available for paternal cellular signaling, we developed an approach to confirm the stress sensitivity and reactivity of experimental mice. We next demonstrated that glucocorticoids are involved in communicating stress to the caput epididymis, a somatic tissue that secretes extracellular vesicles (EVs) to deliver iv microRNA from epididymal epithelial cells to maturing sperm. Using an in vitro model where we administered glucocorticoids to caput epididymal epithelial cells, we showed altered EV microRNA content and within epithelial cells, changes to histone post- translational modifications and increased glucocorticoid receptor levels, mimicking aspects of our in vivo paternal stress model. Further, we demonstrated the crucial role of caput epididymal glucocorticoid receptors in paternal stress transmission by transgenic knockdown, preventing offspring HPA axis and hypothalamic programming. In our final study, we provided evidence for the specificity of paternal stress sperm microRNA effects on embryonic brain and placental transcriptomes, indicating a tightly regulated process by which sperm microRNA are coordinated and function to influence offspring development. Together, the research presented in this dissertation provides insight into the mechanisms contributing to paternal transmission and support the paternal preconception environment as an influential factor in offspring disease risk and resilience. v TABLE OF CONTENTS ACKNOWLEDGEMENT .................................................................................................