SummaryReproductive aging is characterized by a marked decline in oocyte quality that contributes to infertility, miscarriages, and birth defects. This decline is multifactorial, and the underlying mechanisms are under active investigation. Here, we performed RNA‐Seq on individual growing follicles from reproductively young and old mice to identify age‐dependent functions in oocytes. This unbiased approach revealed genes involved in cellular processes known to change with age, including mitochondrial function and meiotic chromosome segregation, but also uncovered previously unappreciated categories of genes related to proteostasis and organelles required for protein metabolism. We further validated our RNA‐Seq data by comparing nucleolar structure and function in oocytes from reproductively young and old mice, as this organelle is central for protein production. We examined key nucleolar markers, including upstream binding transcription factor (UBTF), an RNA polymerase I cofactor, and fibrillarin, an rRNA methyltransferase. In oocytes from mice of advanced reproductive age, UBTF was primarily expressed in giant fibrillar centers (GFCs), structures associated with high levels of rDNA transcription, and fibrillarin expression was increased ~2‐fold. At the ultrastructural level, oocyte nucleoli from reproductively old mice had correspondingly more prominent fibrillar centers and dense fibrillar centers relative to young controls and more ribosomes were found in the cytoplasm. Taken together, our findings are significant because the growing oocyte is one of the most translationally active cells in the body and must accumulate high‐quality maternally derived proteins to support subsequent embryo development. Thus, perturbations in protein metabolism are likely to have a profound impact on gamete health.

Summary Reproductive aging is characterized by a marked decline in oocyte quality that contributes to infertility, miscarriages, and birth defects. This decline is multifactorial, and the underlying mechanisms are under active investigation. Here, we performed RNA‐Seq on individual growing follicles from reproductively young and old mice to identify age‐dependent functions in oocytes. This unbiased approach revealed genes involved in cellular processes known to change with age, including mitochondrial function and meiotic chromosome segregation, but also uncovered previously unappreciated categories of genes related to proteostasis and organelles required for protein metabolism. We further validated our RNA‐Seq data by comparing nucleolar structure and function in oocytes from reproductively young and old mice, as this organelle is central for protein production. We examined key nucleolar markers, including upstream binding transcription factor (UBTF), an RNA polymerase I cofactor, and fibrillarin, an rRNA methyltransferase. In oocytes from mice of advanced reproductive age, UBTF was primarily expressed in giant fibrillar centers (GFCs), structures associated with high levels of rDNA transcription, and fibrillarin expression was increased ~2‐fold. At the ultrastructural level, oocyte nucleoli from reproductively old mice had correspondingly more prominent fibrillar centers and dense fibrillar centers relative to young controls and more ribosomes were found in the cytoplasm. Taken together, our findings are significant because the growing oocyte is one of the most translationally active cells in the body and must accumulate high‐quality maternally derived proteins to support subsequent embryo development. Thus, perturbations in protein metabolism are likely to have a profound impact on gamete health.

The female reproductive system ages before any other organ system in the body. This phenomenon can have tangible clinical implications leading to infertility, miscarriages, birth defects and systemic deterioration due to estrogen loss. “Fibroinflammation” is a hallmark of aging tissues; there is an increase in inflammatory cytokines and fibrotic tissue in the aging ovarian stroma. We systematically evaluated immunomodulatory factors in human follicular fluid, which, like the stroma, is a critical ovarian microenvironment directly influencing the oocyte. Using a cytokine antibody array, we identified a unique fibroinflammatory cytokine signature in follicular fluid across an aging series of women (27.7–44.8 years). This signature (IL-3, IL-7, IL-15, TGFβ1, TGFβ3 and MIP-1) increased with chronologic age, was inversely correlated to anti-Müllerian hormone (AMH) levels, and was independent of body mass index (BMI). We focused on one specific protein, TGFβ3, for further validation. By investigating this cytokine in human cumulus cells and ovarian tissue, we found that the age-dependent increase in TGFβ3 expression was unique to the ovarian stroma but not other ovarian sub-compartments. This study broadens our understanding of inflammaging in the female reproductive system and provides a defined fibroinflammatory aging signature in follicular fluid and molecular targets in the ovary with potential clinical utility.

O‐linked N‐Acetylglucosamine, more commonly referred to as O‐GlcNAc, is a single glycosidic post‐translational modification. OGT (O‐GlcNAc Transferase) and OGA (O‐GlcNAcase) are the sole enzymes responsible for the addition (O‐GlcNAcylation) and removal of O‐GlcNAc, respectively. O‐GlcNAc, OGA, and OGT are present in all multicellular organisms and organ tissues. Importantly, OGT knockouts are embryonically lethal, demonstrating the requirement for O‐GlcNAcylation in early organismal development. Like most intracellular post‐translational modifications, O‐GlcNAc regulates a number of cellular pathways including mitochondrial respiration, cell cycle progression, and transcription. Recently, we demonstrated that prolonged treatment of SY5Y neuroblastoma cells with Thiamet‐G (TMG), an OGA inhibitor, caused a dramatic reprogramming of the transcriptome. Therefore, we performed next generation RNA sequencing to investigate the transcriptome changes from isolated liver tissues from mice treated with TMG for two weeks. We also performed the same analysis for liver tissue containing an OGT knockout (OGT‐KO) to compare the effects of sustained O‐GlcNAcylation to the loss of O‐GlcNAcylation. Gene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed differences in expression for gene sets between the two groups. Further analysis of the data demonstrated an inverse correlation of transcriptional changes for several gene families between TMG‐treated and OGT‐KO. MAPK/ERK signaling genes were among these families, which we found to be of interest. ERK (extracellular regulated protein kinase) phosphorylates a wide variety of protein substrates, regulating processes such as apoptosis, transcription, and cell cycle progression. OGT‐KO was predicted to down‐regulate this pathway while our previous data predicted TMG to up‐regulate the pathway. To test this observation, we grew HeLa cells in TMG for three weeks and serum starved them overnight. The following day, we returned the serum to the media and harvested the lysate at 0, 5,10, 20, 60, and 120 minute time points. When we performed a western blot analysis of the results, the TMG‐treated group demonstrated dramatic amplification of pERK. This observation demonstrates a novel role for O‐GlcNAc as a regulator of the ERK signaling pathway.Support or Funding InformationNIH NIDDK R01 DK100595This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Low-grade endometrial endometrioid adenocarcinomas (LGEECa) can recur in the vagina (VRec), pelvic and abdominal region (PARec), or distant sites (DMet). Tumor size, histopathologic features, and lymph node involvement at presentation have been linked to the development of these recurrences. However, the amount of information on risk factors to predict site of recurrence is limited. Methods: In this multi-institutional study, we analyzed data from 589 patients with FIGO grades 1 and 2 LGEECa and found 116 patients with VRec, PARec, or DMet. They were compared with 187 age-matched controls with negative lymph nodes, no adjuvant treatment, and no recurrences; mean follow-up times were 44 and 59 months, respectively. Cox proportional hazards analysis was used to identify univariable and multivariable risk factors for each type of recurrence (P b .05). Results: Forty-one patients had VRec, 33 had PARec (pelvic soft tissue, 14; abdominal tissue, 9; liver capsule, 5; retroperitoneum, 3; colorectal wall, 2), and 42 had DMet (lung, 19; lymph nodes, 17; bone and soft tissue, 5; brain, 1) as the initial site of recurrence. Univariate and multivariate analysis of histopathologic features are summarized in Table 1. In addition, features associated with vaginaonly recurrence included superficial myometrial invasion (P = .002); low nuclear grade (P = .03); lymphovascular invasion (LVI) adjacent to tumor, but not deeper than invasive tumor front (P b .001); less than 5% microcystic elongated and fragmented pattern (MELF) at invasive tumor front (P = .014); and no pelvic lymph node metastasis at presentation (P = .019). These features were not significantly different from controls. Conclusions: (1) Features of LGEECa that predicted VRec included superficialy invasive, low nuclear grade tumors with minimal MELF, minimal or no LVI, and no lymph node metastasis. These features were more closely related with tumors that did not recur than with recurrent tumors. (2) LGEECa with PARec differed from those with VRec, because the tumors were larger and deeply invasive and MELF at invasive tumor front, suggesting a different dissemination route than tumors with VRec. (3) Significant predictor features of LGEECa with DMet included intraglandular tumor necrosis, cell clusters at the invasive front and adjacent to areas of LVI, and cervical stromal involvement. The latter feature might be indicative of venous rather of lymphatic invasion.