Mechanisms that directly control mammalian ovarian primordial follicle (PF) growth activation and the selection of individual follicles for survival are largely unknown. Follicle cells produce factors that can act as potent inducers of cellular stress during normal function. Consistent with this, we show here that normal, untreated ovarian cells, including pre-granulosa cells of dormant PFs, express phenotype and protein markers of the activated integrated stress response (ISR), including stress-specific protein translation (phospho-Serine 51 eukaryotic initiation factor 2 α ; P-EIF2 α ), active DNA damage checkpoints, and cell cycle arrest. We further demonstrate that mRNAs upregulated in primary (growing) follicles versus arrested PFs mostly include stress-responsive upstream open reading frames (uORFs). Treatment of a granulosa cell (GC) line with the PF growth trigger tumor necrosis factor alpha results in the upregulation of a ‘stress-dependent’ translation profile. This includes further elevated P-eIF2 α and a shift of uORF-containing mRNAs to polysomes. Because the active ISR corresponds to slow follicle growth and PF arrest, we propose that repair and abrogation of ISR checkpoints (e.g. checkpoint recovery) drives the GC cell cycle and PF growth activation (PFGA). If cellular stress is elevated beyond a threshold(s) or, if damage occurs that cannot be repaired, cell and follicle death ensue, consistent with physiological atresia. These data suggest an intrinsic quality control mechanism for immature and growing follicles, where PFGA and subsequent follicle growth and survival depend causally upon ISR resolution, including DNA repair and thus the proof of genomic integrity.

The transcription factor NFκB has been associated with the timing of menopause in a large human genome-wide association study. Furthermore, preclinical studies demonstrate that loss of Tumor necrosis factor alpha (Tnfα) or its receptor Tnfr2 slows primordial follicle growth activation (PFGA). Although Tnfα:receptor signaling stimulates NFκB and may mechanistically link these findings, very little is known about NFκB signaling in PFGA. Because signaling downstream of Tnfα/Tnfr2 ligand/receptor interaction has not been interrogated as relates to PFGA, we evaluated the expression of key NFκB signaling proteins in primordial and growing follicles, as well as during ovarian aging. We show that key members of the NFκB pathway, including subunits, activating kinases and inhibitory proteins, are expressed in the murine ovary. Furthermore, the subunits p65 and p50, and the cytosolic inhibitory proteins IκBα and IκBβ, are present in ovarian follicles, including at the primordial stage. Finally, we assessed PFGA in genetically modified mice (AKBI) previously demonstrated to be resistant to inflammatory stress-induced NFκB activation due to overexpression of the NFκB inhibitory protein IκBβ. Consistent with the hypothesis that NFκB plays a key role in PFGA, AKBI mice exhibit slower PGFA than wild-type (WT) controls, and their ovaries contain nearly twice the number of primordial follicles as WT

We are evaluating the action of the Integrated Stress Response (ISR) pathway and how its enforcement of cell cycle arrest and enhancement of a cytoprotective state contribute to the rate of primordial follicle (PF) loss. Physiological stress and damage are variable locally/regionally within the ovary, thus we predict that ISR activity within PFs will be locations-dependent. We therefore tested whether levels of the marker of the active ISR, serine 51-phosphorylation of eukaryotic Initiation Factor 2 alpha subunit (P-eIF2α), differed between oocytes and pregranulosa cells within PFs, between PFs in the same ovary, and between PFs in ovaries collected from different animals. Quantitative fluorescence analysis revealed that P-eIF2α levels correspond tightly between oocytes and pregranulosa cells within the same PFs, but that different follicles in the same ovary have varied levels of the ISR activity marker; this was consistent between animal replicates. The variation of ISR activity in PFs may therefore directly reflect local/regional differences in physiological factors that induce cell damage and stress. This feature of the dynamically changing intra-ovarian environment may influence the likelihood that PFs stay dormant or begin to grow.

Problem: Immune cell trafficking and surveillance within the ovary and fallopian tube are thought to impact fertility and also tumorigenesis in those organs. However, little is known of how native cells of the ovary and fallopian tube interact with resident immune cells. Interaction of the Programmed Cell Death Protein-1 (PD-1/PDCD-1/CD279) checkpoint with PD-L1 is associated with downregulated immune response.We have begun to address the question of whether PD-1 ligand or its receptors (PD-L1/-L2) can regulate immune cell function in these tissues of the female reproductive tract.Method of study: PD-1 and ligand protein expression was evaluated in human ovary and fallopian tube specimens, the latter of which included stages of tubal cell transformation and early tumorigenesis. Ovarian expression analysis included the determination of the proteins in human follicular fluid (HFF) specimens collected during in vitro fertilization procedures. Finally, checkpoint bioactivity of HFF was determined by treatment of separately-isolated human T cells and the measurement of interferon gamma (IFN𝛾).