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.

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.

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.

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.

Effect of caloric restriction and rapamycin on ovarian aging in mice.

Caloric restriction (CR) increases the preservation of the ovarian primordial follicular reserve, which can potentially delay menopause. Rapamycin also increases preservation on the ovarian reserve, with similar mechanism to CR. Therefore, the aim of our study was to evaluate the effects of rapamycin and CR on metabolism, ovarian reserve, and gene expression in mice. Thirty-six female mice were allocated into three groups: control, rapamycin-treated (4 mg/kg body weight every other day), and 30% CR. Caloric restricted females had lower body weight (P < 0.05) and increased insulin sensitivity (P = 0.003), while rapamycin injection did not change body weight (P > 0.05) and induced insulin resistance (P < 0.05). Both CR and rapamycin females displayed a higher number of primordial follicles (P = 0.02 and 0.04, respectively), fewer primary, secondary, and tertiary follicles (P < 0.05) and displayed increased ovarian Foxo3a gene expression (P < 0.05). Despite the divergent metabolic effects of the CR and rapamycin treatments, females from both groups displayed a similar increase in ovarian reserve, which was associated with higher expression of ovarian Foxo3a.

Effect of serum paraoxonase-1 (PON1) activity on follicular development and pregnancy rate in cattle.

Paraoxonase-1 (PON1) activity has been associated with improvement in ovarian function in early postpartum dairy cows and improved in vitro embryo development. The aim of the current study was to evaluate the potential association among PON1 activity and follicular growth, diameter of the preovulatory follicle and pregnancy per artificial insemination (AI) service in cattle. In Experiment 1, cows (n=33) were subjected to an estradiol-progesterone based protocol to control time of ovulation. Starting on Day 8 of the protocol, follicular growth and serum PON1 activity were monitored. Cows were separated according to the occurrence of ovulation into two groups: Ovulatory (Ov; n=22) and Anovulatory (Anov; n=11). The serum activity of PON1 was not different between Ov and Anov cows (P=0.94). In addition, using a regression model there was no effect of serum PON1 activity on the diameter of dominant follicle (r2=0.00; P=0.99). In Experiment 2, cows (n=193) were submitted to the same hormonal protocol as in Experiment 1. On the day of the timed artificial insemination (TAI), the diameter of dominant follicle was evaluated and blood samples were collected for analysis of PON1 activity. According to the serum PON1 activity, cows were divided into three groups: Low (<70U/mL), Medium (70-90U/mL) or High (>90U/mL) PON1 activity. The overall pregnancy rate was 62.7% (121/193), with no difference among PON1 activity groups. Additionally, using a regression model there was no effect of serum PON1 activity on the diameter of the preovulatory follicle (r2=0.03; P=0.65) and pregnancy rate (r2=0.005; P=0.94). The results of this study indicate that there is no effect of serum PON1 activity on the diameter of preovulatory follicle or establishment of pregnancy in cows submitted to time of ovulation synchronization protocols.