The cycling and aging mouse female reproductive tract at single-cell resolution.

The female reproductive tract (FRT) undergoes extensive remodeling during reproductive cycling. This recurrent remodeling and how it shapes organ-specific aging remains poorly explored. Using single-cell and spatial transcriptomics, we systematically characterized morphological and gene expression changes occurring in ovary, oviduct, uterus, cervix, and vagina at each phase of the mouse estrous cycle, during decidualization, and into aging. These analyses reveal that fibroblasts play central-and highly organ-specific-roles in FRT remodeling by orchestrating extracellular matrix (ECM) reorganization and inflammation. Our results suggest a model wherein recurrent FRT remodeling over reproductive lifespan drives the gradual, age-related development of fibrosis and chronic inflammation. This hypothesis was directly tested using chemical ablation of cycling, which reduced fibrotic accumulation during aging. Our atlas provides extensive detail into how estrus, pregnancy, and aging shape the organs of the female reproductive tract and reveals the unexpected cost of the recurrent remodeling required for reproduction.

microRNA-449a reduces growth hormone-stimulated senescent cell burden through PI3K-mTOR signaling.

Cellular senescence, a hallmark of aging, has been implicated in the pathogenesis of many major age-related disorders, including neurodegeneration, atherosclerosis, and metabolic disease. Therefore, investigating novel methods to reduce or delay the accumulation of senescent cells during aging may attenuate age-related pathologies. microRNA-449a-5p (miR-449a) is a small, noncoding RNA down-regulated with age in normal mice but maintained in long-living growth hormone (GH)-deficient Ames Dwarf (df/df) mice. We found increased fibroadipogenic precursor cells, adipose-derived stem cells, and miR-449a levels in visceral adipose tissue of long-living df/df mice. Gene target analysis and our functional study with miR-449a-5p have revealed its potential as a serotherapeutic. Here, we test the hypothesis that miR-449a reduces cellular senescence by targeting senescence-associated genes induced in response to strong mitogenic signals and other damaging stimuli. We demonstrated that GH downregulates miR-449a expression and accelerates senescence while miR-449a upregulation using mimetics reduces senescence, primarily through targeted reduction of p16Ink4a, p21Cip1, and the PI3K-mTOR signaling pathway. Our results demonstrate that miR-449a is important in modulating key signaling pathways that control cellular senescence and the progression of age-related pathologies.

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.

Metabolic and productive parameters of lactating dairy cows under heat stress conditions supplemented with plant polyphenol extract.

The impact of heat stress on dairy cattle leads to significant economic losses and a negative impact on the welfare of the animals. The objective of this research was to evaluate the effect of the nutritional additive (Thermoplus®) in dairy cows under postpartum heat stress conditions, and its effects on the metabolic profile, production and quality of milk. Eighteen lactating Holstein cows (8 multiparous and ten primiparous), in a free-stall system, with a mean body condition score (BCS) of 3.14 ± 0.05, live weight of 624.55 ± 18, 61 kg, with initial mean days in milk (DIM) of 90 ± 10.11, were selected. The animals were grouped into a control (CG, n = 9) and a treatment (TG, n = 9). Both groups underwent 14 days of diet adaptation, the TG received the basal diet supplemented with 50 g of the additive, once a day, individually, while the control group received only the total diet. Data collection of metabolic and productive parameters were evaluated on days -14 (before adaptation), 1 (after the diet adaptation period), 16, 30, and 44. Milk, blood, and body condition score (BCS) were collected once a day, and heart rate, respiratory rate, and rectal temperature were collected twice a day. Serum concentrations of albumin, calcium, magnesium, glucose, gamma-glutamyl transferase (GGT), beta-hydroxybutyrate (BHBA), non-esterified fatty acids (NEFAs), and paraoxonase-1 (PON-1) were evaluated. In the milk, the percentage of fat, protein, lactose, and total solids were determined in each sampling. Milk yield was measured daily. Humidity and ambient temperature values were collected on the days of the collection every 30 min, from 5:30 am to 5:00 pm, to calculate the temperature-humidity index (THI). Statistical analyzes were performed using the SAS software (version 9.3, SAS Institute Inc., Cary, NC, USA). The THI ranged from 62.22 to 79.47. Our findings showed that when the THI was greater than 72, the animals in the TG were able to maintain milk yield (Odds ratio (OD) = -0.0577,), and the animals in the CG had a greater chance of reducing it (OD = -0.2301). Multiparous cows in the TG had higher milk yield than CG (32.57 ± 0.34 vs 30.50 ± 0.36 kg per day; P = 0.0078) and lower SCC (34.110 ± 6,940 vs 665.50 ± 214.41 cells per ml; P = 0.03), with the same percentages of total solids (P > 0.05). In multiparous metabolic markers, TG when compared CG had higher albumin concentrations (2.50 ± 0.07 vs 2.12 ± 0.07 g/dl; < 0.001), equal PON-1 (P > 0.05), and higher BHBA levels (0.49 ± 0.03 vs 0.39 ± 0.04 mmol/l). Primiparous from the CG had higher concentrations of NEFA (0.18 ± 0.02 mmol/l) than multiparous from the same group (0.09 ± 0.02 mmol/l) P = 0.0265. The use of the plant polyphenol extract in postpartum Holstein cows challenged by heat stress had beneficial effects on the production and health of the mammary gland in multiparous cows without decreasing milk solids. The non-reduction of the activities of the acute phase proteins indicates an immunomodulatory and inflammatory-reducing effect of the product used.

Dwarf mice as models for reproductive ageing research.

Dwarf mice are characterized by extremely long lifespan, delayed ovarian ageing, altered metabolism, lower age-related oxidative damage and cancer incidence rate. Snell dwarf, Ames dwarf and growth hormone receptor knockout mice are three commonly used models. Despite studies focusing on ageing and metabolism, the reproductive features of female dwarf mice have also attracted interest over the last decade. Female Snell and Ames dwarf mice have regular oestrous cycles and ovulation rates, as in normal mice, but with a larger ovarian reserve and delayed ovarian ageing. The primordial follicle reserve in dwarf mice is greater than in normal littermates. Anti-Müllerian hormone (AMH) concentration is seven times higher in Ames dwarf mice than in their normal siblings, and ovarian transcriptomic profiling showed distinctive patterns in older Ames dwarf mice, especially enriched in inflammatory and immune response-related pathways. In addition, microRNA profiles also showed distinctive differences in Ames dwarf mice compared with normal control littermates. This review aims to summarize research progress on dwarf mice as models in the reproductive ageing field. Investigations focusing on the mechanisms of their reserved reproductive ability are much needed and are expected to provide additional molecular biological bases for the clinical practice of reproductive medicine in women.

Expression of genes associated with fertility in the uterus and oviduct of heifers challenged with lipopolysaccharide.

Lipopolysaccharide (LPS) endotoxemia has been negatively associated with fertility. This study aimed to investigate the effect of LPS-induced inflammation on gene expression associated with bovine fertility in the uterus and oviduct. Sixteen healthy heifers were divided into two groups. The LPS group (n = 8) received two intravenous (i.v.) injections of 0.5 µg/kg of body weight of LPS with a 24-h interval, and the control group (n = 8) received two i.v. injections of saline solution with the same interval of time. All the animals had the follicular wave synchronized. Three days after the second injection of LPS, all animals were slaughtered and uterine and oviduct samples were collected. Gene expression associated with inflammatory response, thermal and oxidative stresses, oviduct environment quality, and uterine environment quality was evaluated. Body temperature and leucogram demonstrated that LPS induced an acute systemic inflammatory response. In the uterus, the expression of PTGS2 and NANOG genes was downregulated by the LPS challenge. However, no change in expression was observed in the other evaluated genes in the uterus, nor those evaluated in the oviduct. In conclusion, the inflammatory process triggered by LPS did not persist in the uterus and oviduct 3 days after challenge with LPS. Nonetheless, reduction in PTGS2 and NANOG expression in the uterus suggested that, indirectly, LPS may have a prolonged effect, which may affect corpus luteum and endometrial functions.

Combination of ACTN3 R577X and ACE I/D polymorphisms as a tool for prediction of obesity risk in children.

The genetic influence in obesity prevalence is well described, but the role of genetic markers related to athletic strength/ endurance performance remains controversial. We investigated associations between obesity and the genetic polymorphisms alpha-actinin-3 (ACTN3) R577X and angiotensin-converting enzyme (ACE) I/D in schoolchildren aged 4-13 years from Southern Brazil. We collected sociodemographic data from parents through a questionnaire and conducted an anthropometric assessment. DNA was extracted from buccal cells and genotyping was performed by PCR. We found that 1.9% of the individuals were classified as low weight-for-age, 57.6% as normal weight and 40.5% as overweight/ obesity. Regarding allelic distribution, we found that 52.5% of individuals were DD, 30.8% ID, and 16.7% II for ACE; and 38.8% of individuals were RR, 40.2% RX and 21.0% XX for ACTN3. When both polymorphisms were combined, we observed a clear association between the composed genetic profile of these alleles and severe obesity in schoolchildren. Our data suggest that the combined analysis of ACTN3 R577X and ACE I/D polymorphisms may serve as a predictor for the risk of severe obesity in children. These data can contribute to a better understanding of the relationship between these polymorphisms and the body weight development of school-age children.

Hypolipidemic and anti-inflammatory properties of phenolic rich Butia odorata fruit extract: potential involvement of paraoxonase activity.

Aim: This study investigated the effects of polar Butia odorata fruit extract on metabolic, inflammatory, and oxidative stress parameters in rats submitted to a hyperlipidaemia condition induced by tyloxapol.Methods: Animals were divided into 3 groups: saline, saline plus tyloxapol, and B. odorata extract plus tyloxapol. Animals were treated for 15 days with a saline solution or B. odorata fruit extract and after hyperlipidaemia was induced by tyloxapol.Results: Treatment with B. odorata extract reduced serum triglyceride, total cholesterol, C-reactive protein, and adenosine deaminase and butyrylcholinesterase activities when compared to the tyloxapol group. HDL-cholesterol and paraoxonase 1 activity were higher in B. odorata extract treated animals when compared to tyloxapol-treated animals. No differences were observed in hepatic oxidative stress parameters. Phenolic compounds present in B. odorata fruit extract were identified and quantified by LC-MS/MS.Conclusion: These findings indicated that phenolic rich B. odorata extract has hypolipidemic and anti-inflammatory effects in hyperlipidemic rats.

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.

Supplementation with beta-hydroxy-beta-methylbutyrate impacts glucose homeostasis and increases liver size in trained mice.

ß-hydroxy-ß-methyl butyrate (HMB) is a bioactive metabolite derived from the amino acid leucine, usually applied for muscle mass increase during physical training, as well as for muscle mass maintenance in debilitating chronic diseases. The hypothesis of the present study is that HMB is a safe supplement for muscle mass gain by strength training. Based on this, the objective was to measure changes in body composition, glucose homeostasis and hepatic metabolism of HMB supplemented mice during strength training. Two of four groups of male mice (n = 6/group) underwent an 8-week training period session (climbing stairs) with or without HMB supplementation (190 mg/kgBW per day). We observed lower body mass gain (4.9 ± 0.43% versus 1.2 ± 0.43, p < 0.001) and increased liver mass (40.9 ± 0.9 mg/gBW versus 44.8 ± 1.3, p < 0.001) in the supplemented trained group compared with the non-supplemented groups. The supplemented trained group had an increase in relative adipose tissue mass (12.4 ± 0.63 mg/gBW versus 16.1 ± 0.88, P < 0.01) compared to the non-supplemented untrained group, and an increase in fasting blood glucose (111 ± 4.58 mg/dL versus 122 ± 3.70, P < 0.05) and insulin resistance (3.79 ± 0.19 % glucose decay/min versus 2.45 ± 0.28, P < 0.05) comparing with non-supplemented trained group. Adaptive heart hypertrophy was observed only in the non-supplemented trained group (4.82 ± 0.05 mg/gBW versus 5.12 ± 0.13, P < 0.05). There was a higher hepatic insulin-like growth factor-1 expression (P = 0.002) in supplemented untrained comparing with non-supplemented untrained group. Gene expression of gluconeogenesis regulatory factors was increased by training and reduced by HMB supplementation. These results confirm that HMB supplementation associated with intensive training protocol drives changes in glucose homeostasis and liver metabolism in mice.

Early embryonic development of bovine oocytes challenged with LPS in vitro or in vivo.

The aim of this study was to evaluate the effect of exposing bovine oocytes to lipopolysaccharides (LPS) in vivo and in vitro on early embryo development. In experiment 1, cumulus oocyte complexes (COCs, n = 700/group) were challenged with 0, 0.1, 1.0 or 5.0 µg/mL of LPS during in vitro maturation (IVM). Later, in vitro fertilization (IVF) and in vitro culture (IVC) were performed. In experiment 2, COCs (n = 200/group) matured and in vitro fertilized without LPS were subjected to IVC with the same doses of LPS from experiment 1. In experiment 3, heifers received two injections of saline solution (n = 8) or 0.5 µg/kg of LPS (n = 8) 24 h apart, and 3 days later, COCs were recovered and submitted to IVM, IVF, and IVC. In experiments 1 and 3, the expression of TLR4, TNF, AREG and EREG genes in cumulus cells was evaluated. Exposure to 1 and 5 µg/mL of LPS during IVM decreased nuclear maturation (39.4 and 39.6%, respectively) compared with control (63.6%, P < 0.05). Despite that, no effect on cleavage and blastocyst rates were observed. Exposure to LPS during IVC did not affect embryonic development. In vivo exposure to LPS decreased the in vitro cleavage rate (54.3 vs 70.2%, P = 0.032), but cleaved embryos developed normally. Number of cells per embryo and gene expression were not affected by the LPS challenge in any experiment. In conclusion, although in vitro exposure to LPS did not affect early embryo development, in vivo LPS exposure reduced cleavage rate.

Effect of butafosfan supplementation during oocyte maturation on bovine embryo development.

Around 60-80% of oocytes maturated in vivo reached competence, while the proportion of maturation in vitro is rarely higher than 40%. In this sense, butafosfan has been used in vivo to improve metabolic condition of postpartum cows, and can represent an alternative to increase reproductive efficiency in cows. The aim of this study was to evaluate the addition of increasing doses of butafosfan during oocyte maturation in vitro on the initial embryo development in cattle. In total, 1400 cumulus-oocyte complexes (COCs) were distributed in four groups and maturated according to supplementation with increasing concentrations of butafosfan (0 mg/ml, 0.05 mg/ml, 0.1 mg/ml and 0.2 mg/ml). Then, 20 oocytes per group were collected to evaluate nuclear maturation and gene expression on cumulus cells and oocytes and the remaining oocytes were inseminated and cultured until day 7, when blastocysts were collected for gene expression analysis. A dose-dependent effect of butafosfan was observed, with decrease of cleavage rate and embryo development with higher doses. No difference between groups was observed in maturation rate and expression of genes related to oocyte quality. Our results suggest that butafosfan is prejudicial for oocytes, compromising cleavage and embryo development.

Intrafollicular paraoxonase 1 activity and the steroidogenic potential of the first post-partum dominant follicle in dairy cows.

Cows experiencing high levels of inflammation and specific metabolic conditions tend to have slower follicular growth and lower serum and follicular concentrations of oestradiol (E2). Paraoxonase1 (PON1) activity decreases during inflammatory processes. Therefore, the aim of this study was to evaluate the association between serum and intrafollicular (FF) PON1 activity and the serum and intrafollicular levels of E2 and progesterone (P4), as well as the mRNA expression of genes related to steroidogenesis, metabolism and inflammation in the first post-partum dominant follicle of Holstein cows. No correlation was found between PON1 activity, the expression of the analysed genes and levels of follicular E2 and P4, except for a negative correlation between serum E2 and follicular PO1 activity. Also, no correlation was found between serum and follicular PON1 during the first post-partum follicular wave.

Effect of high-density lipoprotein on oocyte maturation and bovine embryo development in vitro.

High-density lipoprotein (HDL) is the main lipoprotein in the follicular fluid, and it has anti-inflammatory, antioxidant and cryoprotectant properties. The anti-inflammatory potential and antioxidant potential are derived from its lipid composition, especially the apolipoprotein AI (ApoAI) and paraoxonase 1 (PON1). The aim of this study was to evaluate the effect of HDL during in vitro maturation (IVM) on oocyte maturation and early bovine embryo development. For this, cumulus-oocyte complexes (COCs) were obtained from bovine ovaries collected at a local slaughterhouse. COCs (n = 2,250) were allocated into three groups (n = 50 COCs/group) according to the addition of HDL protein (HDL-P) during IVM for 22 hr: 0 (control), 50 and 150 mg/dl. After IVM, COCs were inseminated (in vitro fertilization) and cultivated for 7 days. Total cholesterol concentration, total protein, triglycerides and ApoAI concentrations on IVM medium increased proportionally to HDL-P addition. However, PON1 activity was not detected in any treatment. The addition of HDL-P did not affect nuclear maturation rate, endogenous reactive oxygen species and glutathione levels in COCs (p > 0.05). The highest HDL-P concentration (150 mg/dl) decreased cleavage and blastocyst rate (p < 0.05). Moreover, the HDL-P 150 mg/dl group had lower cellular count/blastocyst than the 50 mg/dl group (p < 0.05). However, the addition of HDL-P did not affect relative gene expression of evaluated genes. In conclusion, the complex HDL/ApoAI obtained from human plasma, in the absence of PON1 activity during in vitro oocyte maturation, decreased initial embryo development.

Multivariate analysis as an advantageous approach for prediction of the adverse outcome in head and neck microvascular reconstructive surgery.

BACKGROUND: The use of a free flap has become a mainstay of reconstruction following the ablative surgery in head and neck. The success rates are about 90%, however, several factors have been described to have an adverse effect on free flap survival. METHODS: We have performed a retrospective analysis of the treatment outcome of 93 microvascular flaps and evaluated the factors influencing the risk of flap loss including patients' age, body mass index, smoking, general medical history and previous oncological treatment. RESULTS: Out of 93 flaps the total necrosis have been observed in 15 flaps with gradual improvement in the consecutive years. In individual analysis the patients age, BMI, and comorbidities did not reveal any significant relation. The history of any previous oncological treatment represented a significant adverse factor of success rate (p=0.035), and was even more significant when patients experienced all treatment modalities prior to the reconstructive procedure (p=0.009). Multivariate logistic regression model indicated that only surgery (p=0.0008), chemotherapy (p=0.02), cardiovascular diseases (p=0.05) and patient's age (p=0.02) represented significant factors impairing the success rate. CONCLUSION: Incorporating multivariate analysis represents important statistical approach for better prediction of free flaps survival in head and neck reconstructive surgery. Incorporation of additional collective information could provide more precise approach in the risk of the flap loss assessment.

Is immunohistochemical evaluation of p16 in oropharyngeal cancer enough to predict the HPV positivity?

AIM: Our goal was to determine the expression levels of p16 in the cohort of the OPSCC patients and evaluation of the pathological and clinical differences between these two groups including patients' survival. BACKGROUND: HPV infection is the main causative factor of oropharyngeal cancer (OPSCC). Identification of HPV status in OPSCC requires positive evaluation of viral DNA integration into host cell however, p16 accumulation in the proliferating cell layers has been accepted as an alternative marker for HPV infection. MATERIAL AND METHODS: The IHC staining for p16 has been performed in tumor tissue from 382 OPSCC patients. The sample was considered positive based on more than 70% of carcinoma tissue showing strong and diffused nuclear and cytoplasmic immunostaining. The clinicopathological characteristics of the patients including site, age, gender, tumor grade, tumor stage, the nodal status, smoking and survival have been analyzed when comparing p16 positive and p16 negative tumors. RESULTS: Out of our cohort in 38.2% cases positive staining for p16 has been recorded. Our analysis did not indicate significant differences in the distribution of the p16 positive patients and age of the patients, stage of the disease. Among the patients who have presented with the N+ neck, there were significantly more p16 positive tumors than in the group with N0 neck (p = 0.0062). There was highly significant correlation between the expression of p16 and smoking (p < 0.0001). The significant difference in survival (p < 0.0001) with more favorable prognosis in the p16 positive group has been observed. CONCLUSIONS: Overexpression of p16 is accepted as a surrogate diagnostic marker for detecting HPV infection in oropharyngeal cancer. However, one should remember about existence of the small subgroups of p16 positive but HPV negative tumors, with relatively worse prognosis. Immunostaining for p16, however useful on everyday basis, should be complemented with other techniques in terms of reliable identification of the HPV infection.

Differential effects of a high-fat diet on serum lipid parameters and ovarian gene expression in young and aged female mice.

The aim of this study was to compare serum lipid profiles and ovarian gene expression between aged and younger female mice fed a control or a high-fat diet for 2 months. For this 16 female mice (C57BL/6) of 4 months (Young, n = 8) or 13 months (Old, n = 8) of age were used. The females were divided into four groups: (i) young females fed a normal diet; (ii) young females fed a high-fat diet; (iii) old females fed a normal diet; and (iv) old females fed a high-fat diet. Food intake was reduced (P < 0.05) in mice fed with a high-fat (2.9 ± 0.1 g) diet in comparison with control mice (3.9 ± 0.1 g). Body weight was higher for old females on the high-fat diet (35.1 ± 0.3 g) than for young females on the same diet (23.3 ± 0.4 g; P < 0.05). PON1 activity was lower in the high-fat than control diet group (114.3 ± 5.8 vs. 78.1 ± 6.0 kU/L, respectively) and was higher in older than younger females (85.9 ± 6.4 vs. 106.5 ± 5.3; P < 0.05, respectively). Females fed a high-fat diet had lower expression of Igf1 mRNA (P = 0.04). There was an interaction between age and diet for the expression of Gdf9 and Survivin, with lower expression in older females in both diets and young females that received the high-fat diet (P < 0.05). Concluding, the high-fat diet reduced the expression of ovarian Igf1 mRNA, and Gdf9 and Survivin mRNA in younger females, which can indicate lower fertility rates. High-density lipoprotein concentration and PON1 activity were higher in aged female mice.

Chronological and reproductive aging-associated changes in resistance to oxidative stress in post-reproductive female mice.

Effort toward reproduction is often thought to negatively influence health and survival. Reproduction has been shown to influence metabolism, but the pathways and mechanisms have yet to be thoroughly elucidated. In the current experiments, our aim was to dissect the role of young and old ovarian tissues in the response to oxidative stress, through changes in liver oxidative stress response proteins. Liver proteins were analyzed in control mice at 4, 13, and 27 months of age and compared to 23-month-old mice which received young ovarian tissue transplants (intact or follicle-depleted) at 13 months of age. In control mice, of the 29 oxidative stress response proteins measured, 31% of the proteins decreased, 52% increased, and 17% were unchanged from 13 to 27 months. The greatest changes were seen during the period of reproductive failure, from 4 to 13 months of age. In transplanted mice, far more proteins were decreased from 13 to 23 months (93% in follicle-containing young ovary recipients; 62% in follicle-depleted young ovary recipients). Neither transplant group reflected changes seen in control mice between 13 and 27 months. Estradiol levels in transplant recipient mice were not increased compared with age-matched control mice. The current results suggest the presence of a germ cell- and estradiol-independent ovarian influence on aging-associated changes in the response to oxidative stress, which is manifest differently in reproductive-aged adults and post-reproductive-aged mice. The results presented here separate chronological and ovarian aging and the influence of estradiol in the response to aging-associated oxidative stress and support a novel, estradiol-independent role for the ovary in female health and survival.

Calorie restriction and life-extending mutation downregulate miR-34a to facilitate lipid metabolism in the liver.

Ames dwarf mice (df/df) display delayed aging relative to their normal (N) siblings, living approximately 40-60 % longer. As such, investigating the mechanisms that enable these organisms to have extended lifespan is useful for the development of interventions to slow aging and deter age-related disease. Nonalcoholic fatty liver disease (NAFLD) is a condition that is characterized by the accumulation of excess adipose tissue in the liver. Previous studies highlight the potential of calorie restriction (CR) in promoting longevity, but little is known about its effects on the biomolecular processes that govern NAFLD. In this study, we examined the role of 6-month CR on genes regulating lipid metabolism in the livers of long-living df/df mice and their N littermates. Importantly, our findings showed significant downregulation of miR-34a-5p in N-CR mice and df/df mice regardless of dietary regimen. Alongside, our RT-PCR results indicated that downregulation of miR-34a-5p is correlated with the expression of metabolism-associated mRNAs involved in modulating the processes of de novo lipogenesis (DNL), fatty acid oxidation (FAO), very-low density lipoprotein transport (VLDL-T), and reverse cholesterol transport (RCT). To further verify the role of miR-34a-5p in regulating metabolic processes, we transfected the human liver cancer (HepG2) cell line with miR-34a mimic, and studied its effect on direct targets Sirt1, Ampk, and Ppara as well as downstream lipid transport regulating genes. Our findings suggest that CR and df/df life extending mutation are robust drivers of the miR-34a-5p signaling pathway and prevent the pathogenesis of age-related diseases by improving overall lipid homeostasis.

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.