MASLD does not affect fertility and senolytics fail to prevent MASLD progression in male mice.

Senescent cells have been linked to the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD). However, the effectiveness of senolytic drugs in reducing liver damage in mice with MASLD is not clear. Additionally, MASLD has been reported to adversely affect male reproductive function. Therefore, this study aimed to evaluate the protective effect of senolytic drugs on liver damage and fertility in male mice with MASLD. Three-month-old male mice were fed a standard diet (SD) or a choline-deficient western diet (WD) until 9 months of age. At 6 months of age mice were randomized within dietary treatment groups into senolytic (dasatinib + quercetin [D + Q]; fisetin [FIS]) or vehicle control treatment groups. We found that mice fed choline-deficient WD had liver damage characteristic of MASLD, with increased liver size, triglycerides accumulation, fibrosis, along increased liver cellular senescence and liver and systemic inflammation. Senolytics were not able to reduce liver damage, senescence and systemic inflammation, suggesting limited efficacy in controlling WD-induced liver damage. Sperm quality and fertility remained unchanged in mice developing MASLD or receiving senolytics. Our data suggest that liver damage and senescence in mice developing MASLD is not reversible by the use of senolytics. Additionally, neither MASLD nor senolytics affected fertility in male mice.

Early life interventions metformin and trodusquemine metabolically reprogram the developing mouse liver through transcriptomic alterations.

Recent studies have demonstrated the remarkable potential of early life intervention strategies at influencing the course of postnatal development, thereby offering exciting possibilities for enhancing longevity and improving overall health. Metformin (MF), an FDA-approved medication for type II diabetes mellitus, has recently gained attention for its promising anti-aging properties, acting as a calorie restriction mimetic, and delaying precocious puberty. Additionally, trodusquemine (MSI-1436), an investigational drug, has been shown to combat obesity and metabolic disorders by inhibiting the enzyme protein tyrosine phosphatase 1b (Ptp1b), consequently reducing hepatic lipogenesis and counteracting insulin and leptin resistance. In this study, we aimed to further explore the effects of these compounds on young, developing mice to uncover biomolecular signatures that are central to liver metabolic processes. We found that MSI-1436 more potently alters mRNA and miRNA expression in the liver compared with MF, with bioinformatic analysis suggesting that cohorts of differentially expressed miRNAs inhibit the action of phosphoinositide 3-kinase (Pi3k), protein kinase B (Akt), and mammalian target of rapamycin (Mtor) to regulate the downstream processes of de novo lipogenesis, fatty acid oxidation, very-low-density lipoprotein transport, and cholesterol biosynthesis and efflux. In summary, our study demonstrates that administering these compounds during the postnatal window metabolically reprograms the liver through induction of potent epigenetic changes in the transcriptome, potentially forestalling the onset of age-related diseases and enhancing longevity. Future studies are necessary to determine the impacts on lifespan and overall quality of life.

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.

Fisetin modulates the gut microbiota alongside biomarkers of senescence and inflammation in a DSS-induced murine model of colitis.

Colitis, a subtype of inflammatory bowel disease (IBD), is a multifactorial disorder characterized by chronic inflammation of the colon. Among various experimental models used in the study of IBD, the chemical colitogenic dextran sulfate sodium (DSS) is most commonly employed to induce colitis in vivo. In the search for new therapeutic strategies, Fisetin, a flavonoid found in many fruits and vegetables, has recently garnered attention for its senolytic properties. Female mice were administered 2.5% DSS in sterile drinking water and were subsequently treated with Fisetin or vehicle by oral gavage. DSS significantly upregulated beta-galactosidase activity in colonic proteins, while Fisetin remarkably inhibited its activity to baseline levels. Particularly, qPCR revealed that the senescence and inflammation markers Vimentin and Ptgs2 were elevated by DSS exposure with Fisetin treatment inhibiting the expression of p53, Bcl2, Cxcl1, and Mcp1, indicating that the treatment reduced senescent cell burden in the DSS targeted intestine. Alongside, senescence and inflammation associated miRNAs miR-149-5p, miR-96-5p, miR-34a-5p, and miR-30e-5p were significantly inhibited by DSS exposure and restored by Fisetin treatment, revealing novel targets for the treatment of IBDs. Metagenomics was implemented to assess impacts on the microbiota, with DSS increasing the prevalence of bacteria in the phyla Bacteroidetes. Meanwhile, Fisetin restored gut health through increased abundance of Akkermansia muciniphila, which is negatively correlated with senescence and inflammation. Our study suggests that Fisetin mitigates DSS-induced colitis by targeting senescence and inflammation and restoring beneficial bacteria in the gut indicating its potential as a therapeutic intervention for IBDs.

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.

Effect of calorie restriction on redox status during chemically induced estropause in female mice.

In females, there is a continuous decline of the ovarian reserve with age, which results in menopause in women or estropause in mice. Loss of ovarian function results in metabolic alterations in mice and women. Based on this, we aimed to evaluate the effect of caloric restriction (CR) on redox status and metabolic changes in chemically induced estropause in mice. For this, mice were divided into four groups (n = 10): cyclic ad libitum (AL), cyclic 30% CR, AL estropause, and estropause 30% CR. Estropause was induced using 4-vinylcyclohexene diepoxide (VCD) for 20 consecutive days in 2-month-old females. The CR protocol started at 5 months of age and the treatments lasted for 4 months. The CR females gained less body weight than AL females (p < 0.001) and had lower glycemic curves in response to glucose tolerance test (GTT). The AL estropause females had the highest body weight and body fat, despite having lower food intake. However, the estropause females on 30% CR lost the most body weight and had the lowest amount of body fat compared to all groups. The effect of 30% CR on redox status in fat and liver tissue was similar for cyclic and estropause females. Interestingly, estropause decreased ROS in adipose tissue, while increasing it in the liver. No significant effects of CR on redox status were observed. Chemically induced estropause did not influence the response to 30% CR on glucose tolerance and redox status; however, weight loss was exarcebated compared to cyclic females.

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.

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.

Senolytic treatment reverses obesity-mediated senescent cell accumulation in the ovary.

Senescent cells are in a cell cycle arrest state and accumulate with aging and obesity, contributing to a chronic inflammatory state. Treatment with senolytic drugs dasatinib and quercetin (D + Q) can reduce senescent cell burden in several tissues, increasing lifespan. Despite this, there are few reports about senescent cells accumulating in female reproductive tissues. Therefore, the aim of the study was to characterize the ovarian reserve and its relationship with cellular senescence in genetically obese mice (ob/ob). In experiment 1, ob/ob (n = 5) and wild-type (WT) mice (n = 5) at 12 months of age were evaluated. In experiment 2, 2-month-old female ob/ob mice were treated with senolytics (D + Q, n = 6) or placebo (n = 6) during the 4 months. Obese mice had more senescent cells in ovaries, indicated by increased p21 and p16 and lipofuscin staining and macrophage infiltration. Treatment with D + Q significantly reduced senescent cell burden in ovaries of obese mice. Neither obesity nor treatment with D + Q affected the number of ovarian follicles. In conclusion, our data indicate that obesity due to leptin deficiency increases the load of senescent cells in the ovary, which is reduced by treatment by senolytics. However, neither obesity nor D + Q treatment affected the ovarian reserve.

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.

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.

Calorie restriction during gestation affects ovarian reserve in offspring in the mouse.

The aim of this study was to investigate the effect of calorie restriction (CR) during pregnancy in mice on metabolism and ovarian function in the offspring. Pregnant female mice were divided into two groups, a control group and a CR group (n=7 in each). Mice in the CR group were fed 50% of the amount consumed by control females from Day 10 of gestation until delivery. After weaning, the offspring received diet ad libitum until 3 months of age, when ovaries were collected. Ovaries were serially cut and every sixth section was used for follicle counting. Female offspring from CR dams tended to have increased bodyweight compared with offspring from control females (P=0.08). Interestingly, fewer primordial follicles (60% reduction; P=0.001), transitional follicles (P=0.0006) and total follicles (P=0.006) were observed in offspring from CR mothers. The number of primary, secondary and tertiary follicles did not differ between the groups (P>0.05). The CR offspring had fewer DNA double-strand breaks in primary follicle oocytes (P=0.03). In summary, CR during the second half of gestation decreased primordial ovarian follicle reserve in female offspring. These findings suggest that undernutrition during the second half of gestation may decrease the reproductive lifespan of female offspring.

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.