Integrated stress response control of granulosa cell translation and proliferation during normal ovarian follicle development

Abstract

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.

Recapitulating Human Ovarian Aging Using Random Walks

“…We have generated immunofluorescence data that shows variable levels of the core ISR regulatory factor P-eIF2 α in mouse PF pregranulosa cells and oocytes (Hagen-Lillevik, submitted, preprint available at https://www.researchsquare.com/article/rs-1682172/v1). However, despite our prior detection of P-eIF2 α in human nongrowing follicles (20), a study comparing P-eIF2 α levels in nongrowing follicles in multiple replicate human ovary specimens has not yet been performed. While it is highly likely that regional differences in stress and damage occur similarly between the mouse and human ovary, it may be that mechanisms that respond to these dynamic local conditions differ between the species.…”

“…We reasoned that if the biological mechanisms at work in individual PFs that dictate the probabilities of arrest or growth over time can be clarified, we might be able to predict and simulate PFGA over time in a way that recapitulates the natural pattern. Our ISR data and proposed model (20) suggested that individual PFs experiencing regionally fluctuating stress and damage (and thus ISR activity) over time might reflect a situation analogous to a random walk (RW; 32, 33) relative to a threshold of growth activation (Fig. ib).…”

“…com/article/rs-1682172/v1). However, despite our prior detection of P-eIF2α in human nongrowing follicles (20), a study comparing P-eIF2α levels in nongrowing follicles in multiple replicate human ovary specimens has not yet been performed. While it is highly likely that regional differences in stress and damage occur similarly between the mouse and human ovary, it may be that mechanisms that respond to these dynamic local conditions differ between the species.…”

“…Cellular stress and particularly oxidative damage have long been associated with ovarian aging (14)(15)(16)(17), and one of our groups is probing how endogenous physiological stress and damage impact PFs directly. Using a combination of wet laboratory and bioinformatics approaches, we have recently identified the stress-and-damage-regulated Integrated Stress Response (ISR) pathway (18,19) as a new potential key regulator of ovarian aging throughout the ovary and at the level of individual PFs (20). In that work we showed that the PFGA stimulator Tnfα leads to a rapid ISR response, including the upregulation of the translation of proteins involved in cellular repair.…”

“…Based upon this, we have developed a model (summarized in the Graphical Abstract, Figure i) based upon physiological regional fluctuations (Fig. ia) of ISR activity that occur in granulosa cells during normal ovarian function (20). We hypothesize that it is checkpoint resolution of physiological stress and DNA damage that allows a switch to an active cell cycle and PFGA (21)(22)(23)(24)(25).…”