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.

Background Preterm birth and respiratory support with invasive mechanical ventilation frequently leads to bronchopulmonary dysplasia (BPD). A hallmark feature of BPD is alveolar simplification. For our preterm lamb model of BPD, invasive mechanical ventilation is associated with postnatal feeding intolerance (reduced nutrition) and sedation. In contrast, preterm lambs managed by non-invasive support (NIS) have normal alveolar formation, appropriate postnatal nutrition, and require little sedation. We used the latter, positive-outcome group to discriminate the contribution of reduced nutrition versus sedation on alveolar simplification. We hypothesized that, restricted nutrition, but not sedation, contributes to impaired indices of alveolar formation in preterm lambs managed by NIS. Methods Preterm lambs managed by NIS for 21d were randomized into three groups: NIS control, NIS plus restricted nutrition, and NIS plus excess sedation. We quantified morphological and biochemical indices of alveolar formation, as well as mesenchymal cell apoptosis and proliferation. Results Restricted nutrition impaired morphological and biochemical indices of alveolar formation, and reduced mesenchymal cell apoptosis and proliferation. Excess sedation did not alter these indices, although mesenchymal cell apoptosis was less. Conclusions Our results demonstrate that restricted nutrition, but not excess sedation, contributes to impaired alveolar formation during the evolution of BPD in chronically ventilated preterm lambs.

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.

Classic Galactosemia is an inborn error of carbohydrate metabolism associated with early-onset primary ovarian insufficiency (POI) in young women. Our understanding of the consequences of galactosemia upon fertility and fecundity of affected women is expanding, but there are important remaining gaps in our knowledge and tools for management, and a need for continued dialogue so that the special features of the condition can be better managed. Here, we review galactosemic POI and its reproductive endocrinological clinical sequelae and summarize current best clinical practices for its management. Special consideration is given to the very early-onset nature of the condition in the pediatric/adolescent patient. Afterward, we summarize our current understanding of the reproductive pathophysiology of galactosemia, including the potential action of toxic galactose metabolites upon the ovary. Our work establishing that ovarian cellular stress reminiscent of endoplasmic reticulum (ER) stress is present in a mouse model of galactosemia, as well as work by other groups, are summarized.

Classic Galactosemia (CG) is a devastating inborn error of the metabolism caused by mutations in the GALT gene encoding the enzyme galactose-1 phosphate uridylyltransferase in galactose metabolism. Severe complications of CG include neurological impairments, growth restriction, cognitive delays, and, for most females, primary ovarian insufficiency. The absence of the GALT enzyme leads to an accumulation of aberrant galactose metabolites, which are assumed to be responsible for the sequelae. There is no treatment besides the restriction of dietary galactose, which does not halt the development of the complications; thus, additional treatments are sorely needed. Supplements have been used in other inborn errors of metabolism but are not part of the therapeutic regimen for CG. The goal of this study was to test two generally recognized as safe supplements (purple sweet potato color (PSPC) and myo-inositol (MI)) that may impact cellular pathways contributing to the complications in CG. Our group uses a GalT gene-trapped mouse model to study the pathophysiology in CG, which phenocopy many of the complications. Here we report the ability of PSPC to ameliorate dysregulation in the ovary, brain, and liver of our mutant mice as well as positive results of MI supplementation in the ovary and brain.