Reproductive Ageing: Inflammation, immune cells, and cellular senescence in the aging ovary.

In brief: Recent reports suggest a relationship between ovarian inflammation and functional declines, although it remains unresolved if ovarian inflammation is the cause or consequence of ovarian aging. In this review, we compile the available literature in this area and point to several current knowledge gaps that should be addressed through future studies. Abstract: Ovarian aging results in reduced fertility, disrupted endocrine signaling, and an increased burden of chronic diseases. The factors contributing to the natural decline of ovarian follicles throughout reproductive life are not fully understood. Nevertheless, local inflammation may play an important role in driving ovarian aging. Inflammation progressively rises in aged ovaries during the reproductive window, potentially affecting fertility. In addition to inflammatory markers, recent studies show an accumulation of specific immune cell populations in aging ovaries, particularly lymphocytes. Other hallmarks of the aging ovary include the formation and accumulation of multinucleated giant cells, increased collagen deposition, and increased markers of cellular senescence. Collectively, these changes significantly impact the quantity and quality of ovarian follicles and oocytes. This review explores recent literature on the alterations associated with inflammation, fibrosis, cell senescence, and the accumulation of immune cells in the aging ovary.

17α-estradiol, a lifespan-extending compound, attenuates liver fibrosis by modulating collagen turnover rates in male mice.

Estrogen signaling is protective against chronic liver diseases, although men and a subset of women are contraindicated for chronic treatment with 17ß-estradiol (17ß-E2) or combination hormone replacement therapies. We sought to determine if 17α-estradiol (17α-E2), a naturally occurring diastereomer of 17ß-E2, could attenuate liver fibrosis. We evaluated the effects of 17α-E2 treatment on collagen synthesis and degradation rates using tracer-based labeling approaches in male mice subjected to carbon tetrachloride (CCl4)-induced liver fibrosis. We also assessed the effects of 17α-E2 on markers of hepatic stellate cell (HSC) activation, collagen cross-linking, collagen degradation, and liver macrophage content and polarity. We found that 17α-E2 significantly reduced collagen synthesis rates and increased collagen degradation rates, which was mirrored by declines in transforming growth factor ß1 (TGF-ß1) and lysyl oxidase-like 2 (LOXL2) protein content in liver. These improvements were associated with increased matrix metalloproteinase 2 (MMP2) activity and suppressed stearoyl-coenzyme A desaturase 1 (SCD1) protein levels, the latter of which has been linked to the resolution of liver fibrosis. We also found that 17α-E2 increased liver fetuin-A protein, a strong inhibitor of TGF-ß1 signaling, and reduced proinflammatory macrophage activation and cytokines expression in the liver. We conclude that 17α-E2 reduces fibrotic burden by suppressing HSC activation and enhancing collagen degradation mechanisms. Future studies will be needed to determine if 17α-E2 acts directly in hepatocytes, HSCs, and/or immune cells to elicit these benefits.

Senolytic treatment fails to improve ovarian reserve or fertility in female mice.

Senescent cell number increases with age in different tissues, leading to greater senescent cell load, proinflammatory stress, and tissue dysfunction. In the current study, we tested the efficacy of senolytic drugs to reduce ovarian senescence and improve fertility in reproductive age female mice. In the first experiment, 1-month-old C57BL/6 female mice were treated every other week with D + Q (n = 24) or placebo (n = 24). At 3 and 6 months of age, female mice were mated with untreated males to evaluate pregnancy rate and litter size. In the second experiment, 6-month-old C57BL/6 female mice were treated monthly with D + Q (n = 30), fisetin (n = 30), or placebo (n = 30). Females were treated once a month until 11 months of age, then they were mated with untreated males for 30 days to evaluate pregnancy rate and litter size. In the first experiment, D + Q treatment did not affect pregnancy rate (P = 0.68), litter size (P = 0.58), or ovarian reserve (P > 0.05). Lipofuscin staining was lower in females treated with D + Q (P = 0.04), but expression of senescence genes in ovaries was similar. In the second experiment, D + Q or fisetin treatment also did not affect pregnancy rate (P = 0.37), litter size (P = 0.20), or ovarian reserve (P > 0.05). Lipofuscin staining (P = 0.008) and macrophage infiltration (P = 0.002) was lower in fisetin treated females. Overall, treatment with D + Q or fisetin did not affect ovarian reserve or fertility but did decrease some senescence markers in the ovary.

A single-cell atlas of the aging mouse ovary.

Ovarian aging leads to diminished fertility, dysregulated endocrine signaling and increased chronic disease burden. These effects begin to emerge long before follicular exhaustion. Female humans experience a sharp decline in fertility around 35 years of age, which corresponds to declines in oocyte quality. Despite a growing body of work, the field lacks a comprehensive cellular map of the transcriptomic changes in the aging mouse ovary to identify early drivers of ovarian decline. To fill this gap we performed single-cell RNA sequencing on ovarian tissue from young (3-month-old) and reproductively aged (9-month-old) mice. Our analysis revealed a doubling of immune cells in the aged ovary, with lymphocyte proportions increasing the most, which was confirmed by flow cytometry. We also found an age-related downregulation of collagenase pathways in stromal fibroblasts, which corresponds to rises in ovarian fibrosis. Follicular cells displayed stress-response, immunogenic and fibrotic signaling pathway inductions with aging. This report provides critical insights into mechanisms responsible for ovarian aging phenotypes. The data can be explored interactively via a Shiny-based web application.

Effects of calorie, protein, and branched chain amino acid restriction on ovarian aging in mice.

Calorie restriction (CR) is an intervention that promotes longevity and preserves the ovarian reserve. Some studies have observed that the positive impacts of CR can be linked to restriction of protein (PR) and branched-chain amino acids (BCAAs) independent of calorie intake. The aim of this study was to compare the effects of protein and BCAA restriction to 30% CR on the ovarian reserve of female mice. For this, 3 month-old C57BL/6 female mice (n = 35) were randomized into four groups for four months dietary interventions including: control group (CTL; n = 8), 30% CR (CR; n = 9), protein restriction (PR; n = 9) and BCAA restriction (BCAAR; n = 9). Body mass gain, body composition, food intake, serum levels of BCAAs, ovarian reserve and estrous cyclicity were evaluated. We observed that CR, protein and BCAA restriction prevented weight gain and changed body composition compared to the CTL group. The BCAA restriction did not affect the ovarian reserve, while both PR and CR prevented activation of primordial follicles. This prevention occurred in PR group despite the lack of reduction of calorie intake compared to CTL group, and CR did not reduce protein intake in levels similar to the PR group. BCAA restriction resulted in increased calorie intake compared to CTL and PR mice, but only PR reduced serum BCAA levels compared to the CTL group. Our data indicates that PR has similar effects to CR on the ovarian reserve, whereas BCAA restriction alone did not affect it.

The retrotransposon - derived capsid genes PNMA1 and PNMA4 maintain reproductive capacity.

The human genome contains 24 gag -like capsid genes derived from deactivated retrotransposons conserved among eutherians. Although some of their encoded proteins retain the ability to form capsids and even transfer cargo, their fitness benefit has remained elusive. Here we show that the gag -like genes PNMA1 and PNMA4 support reproductive capacity. Six-week-old mice lacking either Pnma1 or Pnma4 are indistinguishable from wild-type littermates, but by six months the mutant mice become prematurely subfertile, with precipitous drops in sex hormone levels, gonadal atrophy, and abdominal obesity; overall they produce markedly fewer offspring than controls. Analysis of donated human ovaries shows that expression of both genes declines normally with aging, while several PNMA1 and PNMA4 variants identified in genome-wide association studies are causally associated with low testosterone, altered puberty onset, or obesity. These findings expand our understanding of factors that maintain human reproductive health and lend insight into the domestication of retrotransposon-derived genes.

Role of Estrogen Receptor α in Aging and Chronic Disease.

Estrogen receptor alpha (ERα) plays a crucial role in reproductive function in both sexes. It also mediates cellular responses to estrogens in multiple nonreproductive organ systems, many of which regulate systemic metabolic homeostasis and inflammatory processes in mammals. The loss of estrogens and/or ERα agonism during aging is associated with the emergence of several comorbid conditions, particularly in females undergoing the menopausal transition. Emerging data also suggests that male mammals likely benefit from ERα agonism if done in a way that circumvents feminizing characteristics. This has led us, and others, to speculate that tissue-specific ERα agonism may hold therapeutic potential for curtailing aging and chronic disease burden in males and females that are at high-risk of cancer and/or cardiovascular events with traditional estrogen replacement therapies. In this mini-review, we emphasize the role of ERα in the brain and liver, summarizing recent evidence that indicates these two organs systems mediate the beneficial effects of estrogens on metabolism and inflammation during aging. We also discuss how 17α-estradiol administration elicits health benefits in an ERα-dependent manner, which provides proof-of-concept that ERα may be a druggable target for attenuating aging and age-related disease burden.

Chromosomal and gonadal factors regulate microglial sex effects in the aging brain.

Biological sex contributes to phenotypic sex effects through genetic (sex chromosomal) and hormonal (gonadal) mechanisms. There are profound sex differences in the prevalence and progression of age-related brain diseases, including neurodegenerative diseases. Inflammation of neural tissue is one of the most consistent age-related phenotypes seen with healthy aging and disease. The pro-inflammatory environment of the aging brain has primarily been attributed to microglial reactivity and adoption of heterogeneous reactive states dependent upon intrinsic (i.e., sex) and extrinsic (i.e., age, disease state) factors. Here, we review sex effects in microglia across the lifespan, explore potential genetic and hormonal molecular mechanisms of microglial sex effects, and discuss currently available models and methods to study sex effects in the aging brain. Despite recent attention to this area, significant further research is needed to mechanistically understand the regulation of microglial sex effects across the lifespan, which may open new avenues for sex informed prevention and treatment strategies.

Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome.

Assessing cell-type-specific epigenomic and transcriptomic changes are key to understanding ovarian aging. To this end, the optimization of the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method was performed for the subsequent paired interrogation of the cell-specific ovarian transcriptome and epigenome using a novel transgenic NuTRAP mouse model. The expression of the NuTRAP allele is under the control of a floxed STOP cassette and can be targeted to specific ovarian cell types using promoter-specific Cre lines. Since recent studies have implicated ovarian stromal cells in driving premature aging phenotypes, the NuTRAP expression system was targeted to stromal cells using a Cyp17a1-Cre driver. The induction of the NuTRAP construct was specific to ovarian stromal fibroblasts, and sufficient DNA and RNA for sequencing studies were obtained from a single ovary. The NuTRAP model and methods presented here can be used to study any ovarian cell type with an available Cre line.

A single-cell atlas of the aging murine ovary.

Ovarian aging leads to diminished fertility, dysregulated endocrine signaling, and increased chronic disease burden. These effects begin to emerge long before follicular exhaustion. Around 35 years old, women experience a sharp decline in fertility, corresponding to declines in oocyte quality. Despite a growing body of work, the field lacks a comprehensive cellular map of the transcriptomic changes in the aging ovary to identify early drivers of ovarian decline. To fill this gap, we performed single-cell RNA sequencing on ovarian tissue from young (3-month-old) and reproductively aged (9-month-old) mice. Our analysis revealed a doubling of immune cells in the aged ovary, with lymphocyte proportions increasing the most, which was confirmed by flow cytometry. We also found an age-related downregulation of collagenase pathways in stromal fibroblasts, which corresponds to rises in ovarian fibrosis. Follicular cells displayed stress response, immunogenic, and fibrotic signaling pathway inductions with aging. This report raises provides critical insights into mechanisms responsible for ovarian aging phenotypes.

Assessing tolerability and physiological responses to 17α-estradiol administration in male rhesus macaques.

17α-estradiol has recently been shown to extend healthspan and lifespan in male mice through multiple mechanisms. These benefits occur in the absence of significant feminization or deleterious effects on reproductive function, which makes 17α-estradiol a candidate for translation into humans. However, human dosing paradigms for the treatment of aging and chronic disease are yet to be established. Therefore, the goals of the current studies were to assess tolerability of 17α-estradiol treatment, in addition to evaluating metabolic and endocrine responses in male rhesus macaque monkeys during a relatively short treatment period. We found that our dosing regimens (0.30 and 0.20 mg/kg/day) were tolerable as evidenced by a lack of GI distress, changes in blood chemistry or complete blood counts, and unaffected vital signs. We also found that the higher dose did elicit mild benefits on metabolic parameters including body mass, adiposity, and glycosylated hemoglobin. However, both of our 17α-estradiol trial doses elicited significant feminization to include testicular atrophy, increased circulating estrogens, and suppressed circulating androgens and gonadotropins. We suspect that the observed level of feminization results from a saturation of the endogenous conjugation enzymes, thereby promoting a greater concentration of unconjugated 17α-estradiol in serum, which has more biological activity. We also surmise that the elevated level of unconjugated 17α-estradiol was subjected to a greater degree of isomerization to 17ß-estradiol, which is aligned with the sevenfold increase in serum 17ß-estradiol in 17α-estradiol treated animals in our first trial. Future studies in monkeys, and certainly humans, would likely benefit from the development and implementation of 17α-estradiol transdermal patches, which are commonly prescribed in humans and would circumvent potential issues with bolus dosing effects.

17α-estradiol does not adversely affect sperm parameters or fertility in male mice: implications for reproduction-longevity trade-offs.

17α-estradiol (17α-E2) is referred to as a nonfeminizing estrogen that was recently found to extend healthspan and lifespan in male, but not female, mice. Despite an abundance of data indicating that 17α-E2 attenuates several hallmarks of aging in male rodents, very little is known with regard to its effects on feminization and fertility. In these studies, we evaluated the effects of 17α-E2 on several markers of male reproductive health in two independent cohorts of mice. In alignment with our previous reports, chronic 17α-E2 treatment prevented gains in body mass, but did not adversely affect testes mass or seminiferous tubule morphology. We subsequently determined that chronic 17α-E2 treatment also did not alter plasma 17ß-estradiol or estrone concentrations, while mildly increasing plasma testosterone levels. We also determined that chronic 17α-E2 treatment did not alter plasma follicle-stimulating hormone or luteinizing hormone concentrations, which suggests 17α-E2 treatment does not alter gonadotropin-releasing hormone neuronal function. Sperm quantity, morphology, membrane integrity, and various motility measures were also unaffected by chronic 17α-E2 treatment in our studies. Lastly, two different approaches were used to evaluate male fertility in these studies. We found that chronic 17α-E2 treatment did not diminish the ability of male mice to impregnate female mice, or to generate successfully implanted embryos in the uterus. We conclude that chronic treatment with 17α-E2 at the dose most commonly employed in aging research does not adversely affect reproductive fitness in male mice, which suggests 17α-E2 does not extend lifespan or curtail disease parameters through tradeoff effects with reproduction.

Dasatinib and quercetin increase testosterone and sperm concentration in mice.

Cellular senescence is a defense mechanism to arrest proliferation of damaged cells. The number of senescent cells increases with age in different tissues and contributes to the development of age-related diseases. Old mice treated with senolytics drugs, dasatinib and quercetin (D+Q), have reduced senescent cells burden. The aim of this study was to evaluate the effects of D+Q on testicular function and fertility of male mice. Mice (n = 9/group) received D (5 mg kg-1) and Q (50 mg kg-1) via gavage every moth for three consecutive days from 3 to 8 months of age. At 8 months mice were breed with young non-treated females and euthanized. The treatment of male mice with D+Q increased serum testosterone levels and sperm concentration and decreased abnormal sperm morphology. Sperm motility, seminiferous tubule morphometry, testicular gene expression and fertility were not affected by treatment. There was no effect of D+Q treatment in ß-galactosidase activity and in lipofuscin staining in testes. D+Q treatment also did not affect body mass gain and testes mass. In conclusion, D+Q treatment increased serum testosterone levels and sperm concentration and decreased abnormal sperm morphology, however did not affect fertility. Further studies with older mice and different senolytics are necessary to elucidate the effects in the decline of sperm output (quality and quantity) associated with aging.

Mild calorie restriction, but not 17α-estradiol, extends ovarian reserve and fertility in female mice.

Calorie restriction (CR) (25-40%) is the most commonly studied strategy for curtailing age-related disease and has also been found to extend reproductive lifespan in female mice. However, the effects of mild CR (10%), which is sustainable, on ovarian aging has not yet been addressed. 17α-estradiol (17α-E2) is another intervention shown to positively modulate healthspan and lifespan in mice but its effects on female reproduction remain unclear. We evaluated the effects of mild CR (10%) and 17α-E2 treatment on ovarian reserve and female fertility over a 24-week period, and compared these effects with the more commonly employed 30% CR regimen. Both 10% and 30% CR elicited positive effects on the preservation of ovarian reserve, whereas 17α-E2 did not alter parameters associated with ovarian function. Following refeeding, both 10% and 30% increased fertility as evidenced by greater pregnancy rates. In aligned with the ovarian reserve data, 17α-E2 also failed to improve fertility. Collectively, these data indicate that 10% CR is effective in preserving ovarian function and fertility, while 17α-E2 does not appear to have therapeutic potential for delaying ovarian aging.

Cellular hallmarks of aging emerge in the ovary prior to primordial follicle depletion.

Decline in ovarian reserve with advancing age is associated with reduced fertility and the emergence of metabolic disturbances, osteoporosis, and neurodegeneration. Recent studies have provided insight into connections between ovarian insufficiency and systemic aging, although the basic mechanisms that promote ovarian reserve depletion remain unknown. Here, we sought to determine if chronological age is linked to changes in ovarian cellular senescence, transcriptomic, and epigenetic mechanisms in a mouse model. Histological assessments and transcriptional analyses revealed the accumulation of lipofuscin aggresomes and senescence-related transcripts (Cdkn1a, Cdkn2a, Pai-1 and Hmgb1) significantly increased with advancing age. Transcriptomic profiling and pathway analyses following RNA sequencing, revealed an upregulation of genes related to pro-inflammatory stress and cell-cycle inhibition, whereas genes involved in cell-cycle progression were downregulated; which could be indicative of senescent cell accumulation. The emergence of these senescence-related markers preceded the dramatic decline in primordial follicle reserve observed. Whole Genome Oxidative Bisulfite Sequencing (WGoxBS) found no genome-wide or genomic context-specific DNA methylation and hydroxymethylation changes with advancing age. These findings suggest that cellular senescence may contribute to ovarian aging, and thus, declines in ovarian follicular reserve. Cell-type-specific analyses across the reproductive lifespan are needed to fully elucidate the mechanisms that promote ovarian insufficiency.

The Interconnections Between Somatic and Ovarian Aging in Murine Models.

The mammalian female is born with a limited ovarian reserve of primordial follicles. These primordial follicles are slowly activated throughout the reproductive lifecycle, thereby determining lifecycle length. Once primordial follicles are exhausted, women undergo menopause, which is associated with several metabolic perturbations and a higher mortality risk. Long before exhaustion of the reserve, females experience severe declines in fertility and health. As such, significant efforts have been made to unravel the mechanisms that promote ovarian aging and insufficiency. In this review, we explain how long-living murine models can provide insights in the regulation of ovarian aging. There is now overwhelming evidence that most life-span-extending strategies, and long-living mutant models simultaneously delay ovarian aging. Therefore, it appears that the same mechanisms that regulate somatic aging may also be modulating ovarian aging and germ cell exhaustion. We explore several potential contributing mechanisms including insulin resistance, inflammation, and DNA damage-all of which are hallmarks of cellular aging throughout the body including the ovary. These findings are in alignment with the disposable soma theory of aging, which dictates a trade-off between growth, reproduction, and DNA repair. Therefore, delaying ovarian aging will not only increase the fertility window of middle age females, but may also actively prevent menopausal-related decline in systemic health parameters, compressing the period of morbidity in mid-to-late life in females.

17α-Estradiol promotes ovarian aging in growth hormone receptor knockout mice, but not wild-type littermates.

Growth hormone receptor knockout mice (GHRKO) have reduced body size and increased insulin sensitivity. These mice are known for having extended lifespan, healthspan and female reproductive longevity. Seventeen α-estradiol (17α-E2) is reported to increase insulin sensitivity and extend lifespan in male mice, with less robust effects in female mice. The aim of this study was to evaluate the ovarian reserve in wild type and GHRKO mice treated with 17α-E2. The mice were divided into four groups, GHRKO mice receiving a standard chow diet, GHRKO mice treated 17α-E2, wild type mice receiving a standard chow diet and WT mice treated with 17α-E2. 17α-E2 was provided in the diet for four months. IGF1 plasma concentrations and changes in body weight were assessed. Histological slides were prepared from the ovaries and the number of follicles was counted. GHRKO mice receiving the control diet had a greater number of primordial follicles and lower numbers of primary follicles compared to the other groups (p < 0.05). 17α-E2 treatment decreased the number of primordial follicles in GHRKO mice (p < 0.05), however had no effect in wild type mice. Treatment with 17α-E2 had no significant effect on the change in body weight during the experiment (p = 0.75). Plasma IGF1 concentrations were significantly lower in GHRKO mice as compared to wild type. In conclusion, we found that GHRKO mice displayed lesser primordial follicle activation as compared to wild type mice, but this phenotype was reversed by 17α-E2 administration, suggesting that ovarian aging is increased by 17α-E2 in long-living mice with extended reproductive longevity.