Under normal physiological conditions, tissue remodeling in response to injury leads to tissue regeneration without permanent damage. However, if homeostasis between synthesis and degradation of extracellular matrix (ECM) components is altered, fibrosis – or the excess accumulation of ECM – can disrupt tissue architecture and function. Several organs, including the heart, lung, and kidney, exhibit age-associated fibrosis. Here we investigated whether fibrosis underlies aging in the ovary - an organ that ages chronologically before other organs. We used Picrosirius Red (PSR), a connective tissue stain specific for collagen I and III fibers, to evaluate ovarian fibrosis. Using brightfield, epifluorescence, confocal, and polarized light microscopy, we validated the specific staining of highly ordered PSR-stained fibers in the ovary. We next examined ovarian PSR staining in two mouse strains (CD1 and CB6F1) across an aging continuum and found that PSR staining was minimal in ovaries from reproductively young adult animals, increased in distinct foci in animals of mid-to-advanced reproductive age, and was prominent throughout the stroma of the oldest animals. Consistent with fibrosis, there was a reproductive aged-associated increase in ovarian hydroxyproline content. We also observed a unique population of multinucleated macrophage giant cells, which are associated with chronic inflammation, within the ovarian stroma exclusively in reproductively old mice. In fact, several genes central to inflammation had significantly higher levels of expression in ovaries from reproductively old mice relative to young. These results establish fibrosis as an early hallmark of the aging ovarian stroma, and this altered microenvironment may contribute to the age-associated decline in gamete quality.
The semiallogenic fetus is tolerated by the maternal immune system through control of innate and adaptive immune responses. Trophoblast cells secrete nanometer scale membranous particles called exosomes, which have been implicated in modulation of the local and systemic maternal immune system. Here we investigate the possibility that exosomes secreted from the first trimester and term placenta carry HLA-G and B7 family immunomodulators. Confocal microscopy of placental sections revealed intracellular colocalization of B7-H1 with CD63, suggesting that B7-H1 associates with subcellular vesicles that give rise to exosomes. First trimester and term placental explants were then cultured for 24 hours. B7H-1 (CD274), B7-H3 (CD276) and HLA-G5 were abundant in pelleted supernatants of these cultures that contained microparticles and exosomes; the latter, however, was observed only in first trimester pellets and was nearly undetectable in term explant-derived pellets. Further purification of exosomes by sucrose density fractionation confirmed the association of these proteins specifically with exosomes. Finally, culture of purified trophoblast cells in the presence or absence of EGF suggested that despite the absence of HLA-G5 association with term explant-derived exosomes, it is present in exosomes secreted from mononuclear cytotrophoblast cells. Further, differentiation of cytotrophoblast cells reduced the presence of HLA-G5 in secreted exosomes. Together, the results suggest that the immunomodulatory proteins HLA-G5, B7-H1 and B7-H3, are secreted from early and term placenta, and have important implications in the mechanisms by which trophoblast immunomodulators modify the maternal immunological environment.
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.
Loss-of-function mutations in the autoimmune regulator (AIRE) gene are responsible for autoimmune polyglandular syndrome type 1 (APS-1), which commonly manifests as infertility in women. AIRE is a transcriptional regulator that promotes expression of tissue-restricted antigens in the thymus, including antigens specific to the ovary. Thymic expression of ovarian genes under AIRE's control may be critical for preventing ovarian autoimmune disease. Because mice lacking Aire are an important APS-1 model, we examined the reproductive properties of female Aire-deficient (Aire(-/-)) mice. Female Aire(-/-) mice on the BALB/c background were examined for reproductive parameters, including fertility, litter sizes, and ovarian follicular reserves. Although delayed puberty was observed in Aire(-/-) mice, all mice entered puberty and exhibited mating behavior. Only 50% of Aire(-/-) females gave an initial litter, and only 16% were able to produce two litters. Ovarian histopathologic examination revealed that 83% of previously bred females lost all ovarian follicular reserves. Among virgin females, follicular depletion was observed in 25% by 8 wk, and by 20 wk, 50%-60% of mice lost all follicles. This was associated with elevated serum follicle-stimulating hormone level and ovarian infiltration of proliferating CD3+ T lymphocytes. Ovulation rates of 6-wk-old Aire(-/-) mice were reduced by 22%, but this difference was not statistically significant. Finally, transplantation experiments revealed that follicular loss depended on factors extrinsic to the ovary. These results suggest that immune-mediated ovarian follicular depletion is a mechanism of infertility in Aire(-/-) mice. The results have important implications in the pathogenesis of ovarian autoimmune disease in women.
Hepatitis C virus (HCV) is the world’s most common blood-borne viral infection for which there is no vaccine. The rates of vertical transmission range between 3–6% with odds 90% higher in the presence of HIV co-infection. Prevention of vertical transmission is not possible due to lack of an approved therapy for use in pregnancy or an effective vaccine. Recently, HCV has been identified as an independent risk factor for pre-term delivery, perinatal mortality and other complications. In this study, we characterized the immune responses that contribute to the control of viral infection at the maternal-fetal interface (MFI) in the early gestational stages. Here we show that primary human trophoblast cells and an extravillous trophoblast cell line (HTR8), from first and second trimester of pregnancy, express receptors relevant for HCV binding/entry and are permissive for HCV-uptake. We found that HCV-RNA sensing by human trophoblast cells induces robust up-regulation of Type I/III IFNs and secretion of multiple chemokines that elicit recruitment and activation of decidual NK cells. Furthermore, we observed that HCV-RNA transfection induces a pro-apoptotic response within HTR8 that could affect the morphology of the placenta. For the first time, we demonstrate that HCV-RNA sensing by human trophoblast cells elicits a strong antiviral response that alters the recruitment and activation of innate immune cells at the MFI. This work provides a paradigm shift in our understanding of HCV-specific immunity at the MFI, as well as novel insights into mechanisms that limit vertical transmission, but may paradoxically lead to virus-related pregnancy complications.
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