The female reproductive system is an instructive model for studying aging mechanisms because it ages decades prior to other organs in the human body. Reproductive function begins to decline when women are only in their mid-30s and ceases completely at menopause. Female reproductive aging phenotypes include reduced endocrine function and decreased gamete quantity and quality. Together, these changes contribute to adverse fertility and general health outcomes, and such consequences are becoming more tangible as medical advances are extending lifespan and women worldwide are delaying childbearing. Several fundamental hallmarks of aging tissues have been identified including impaired protein homeostasis or proteostasis. Proteins are essential for the structure and function of all tissues and are involved in critical cellular processes. As such, regulatory mechanisms are in place to ensure that proteins are synthesized, folded, and modified properly. As proteins age, however, they accumulate various types of damage, and damaged proteins are typically turned over and replaced with newly synthesized functional versions to maintain proteostasis. Most proteins last only a total of two days or less. However, a unique class of proteins called extremely long lived proteins (ELLPs) can last the entire lifetime of an organism without being replaced. ELLPs are typically part of large complexes (e.g. histones, nuclear pores, structural networks) and underpin aging because accumulated damage compromises their function and may also elicit abnormal signaling pathways. The pathogenic properties of ELLPs are particularly problematic in post-mitotic cells because they are not diluted through cell division. In fact, neuronal ELLPs are implicated in aging and neurodegenerative conditions. Like neurons, the mammalian oocyte is also vulnerable to ELLP dysfunction because it is non-dividing but rather maintained in an extended prophase I arrest for up to months in mouse and decades in human. Thus, damaged ELLPs could accumulate, reduce gamete quality, and may even be passed onto the next generation through the embryo. While ELLPs provide a compelling intellectual framework for considering mechanisms of female reproductive aging, their identification and quantification at the single protein level has been historically challenging due to technical limitations especially within limited biological material. Here we propose to combine a two generation whole animal stable pulse- chase isotope labelling approach with advanced mass spectrometry-based approaches to identify and quantify the extremely long lived proteome in the ovary and oocyte (Aim 1) and to image and quantitatively analyze long lived molecules (proteins, lipids, nucleotides) in the oocyte and ovary within the context of the in vivo microenvironment (Aim 2). These experiments will be performed at two time points across the reproductive aging continuum and encompass the necessary pioneering steps to identify specific extremely long lived substrates and structures within the mammalian oocyte and ovary. The discoveries made here will lay the foundation for future mechanistic studies and have implications not only for reproductive aging but aging tissues more broadly.
Aging is associated with cellular and tissue deterioration and is a prime risk factor for chronic diseases and declining health, with the female reproductive system being the first to show overt manifestations of aging (i.e. decreased fertility, miscarriages, and birth defects). A key hallmark of aging in many tissues is loss of protein homeostasis or quality control mechanisms that protect the cellular proteome. Our research promises to open new avenues of discovery through the use of state-of-the-art mass spectrometry and imaging approaches to identify, visualize, and quantify a unique population of extremely long lived proteins in the oocyte and ovary that persist through the entire reproductive lifespan and likely fuel reproductive aging through accumulated damage.
