FOXO3a deregulation in uterine smooth muscle tumors.

OBJECTIVE: The present study aimed to investigate FOXO3a deregulation in Uterine Smooth Muscle Tumors (USMT) and its potential association with cancer development and prognosis. METHODS: The authors analyzed gene and protein expression profiles of FOXO3a in 56 uterine Leiomyosarcomas (LMS), 119 leiomyomas (comprising conventional and unusual leiomyomas), and 20 Myometrium (MM) samples. The authors used techniques such as Immunohistochemistry (IHC), FISH/CISH, and qRT-PCR for the present analyses. Additionally, the authors conducted an in-silico analysis to understand the interaction network involving FOXO3a and its correlated genes. RESULTS: This investigation revealed distinct expression patterns of the FOXO3a gene and protein, including both normal and phosphorylated forms. Expression levels were notably elevated in LMS, and Unusual Leiomyomas (ULM) compared to conventional Leiomyomas (LM) and Myometrium (MM) samples. This upregulation was significantly associated with metastasis and Overall Survival (OS) in LMS patients. Intriguingly, FOXO3a deregulation did not seem to be influenced by EGF/HER-2 signaling, as there were minimal levels of EGF and VEGF expression detected, and HER-2 and EGFR were negative in the analyzed samples. In the examination of miRNAs, the authors observed upregulation of miR-96-5p and miR-155-5p, which are known negative regulators of FOXO3a, in LMS samples. Conversely, the tumor suppressor miR-let7c-5p was downregulated. CONCLUSIONS: In summary, the outcomes of the present study suggest that the imbalance in FOXO3a within Uterine Smooth Muscle Tumors might arise from both protein phosphorylation and miRNA activity. FOXO3a could emerge as a promising therapeutic target for individuals with Unusual Leiomyomas and Leiomyosarcomas (ULM and LMS), offering novel directions for treatment strategies.

FOXO3 and PTEN expression in the ovary of girls with extra-gonadal cancer with or without chemotherapy treatment prior to cryopreservation.

BACKGROUND: FOXO3/pFOXO3 and PTEN expression is known to regulate the dormancy/activation of ovarian primordial follicles. How chemotherapy could influence the expression of FOXO3 and PTEN in pre- and post-menarcheal girls with extra-gonadal cancer remains unexplored. METHODS: Ovarian samples were collected from 27 girls suffering from extra-gonadal cancer. Of these, 8 patients had received chemotherapy before the time of sample collection. Ovarian tissue collected at the time of surgery was fixed in 10% formaldehyde for FOXO3/pFOXO3 and PTEN immunohistochemistry or immunofluorescence, or stored at -80 °C for Western blot, or preserved in RNA later for RT-PCR. RESULTS: PTEN was detected in a limited number of primordial follicle-enclosed oocytes in approximately fifty percent of the patients, regardless of whether they had received anti-cancer treatment or not. However, there was a significant decrease in PTEN detection in patients who underwent chemotherapy treatment prior to the retrieval of the sample. Both primordial follicle-enclosed oocytes that expressed FOXO3 and those that did not were identified in patients who were treated with chemotherapy and those who were not. FOXO3-positive primordial follicles exhibited either nuclear FOXO3 localization or cytoplasmic pFOXO3 localization. Furthermore, transitional primordial follicles that expressed nuclear FOXO3 and cytoplasmic pFOXO3 were also observed. Primary follicle-enclosed oocytes displayed cytoplasmic pFOXO3 localization, whereas in more advanced stages of folliculogenesis, the expression moved to the somatic stratum. No significant statistical differences were identified in the detection of FOXO3 and pFOXO3 in patients who had or had not received chemotherapy prior to sample collection. CONCLUSION: Primordial follicles expressing and not expressing FOXO3 were equally present in both the ovaries of patients who underwent chemotherapy and those who did not. The expression of FOXO3 remained unaltered in response to chemotherapy treatment. Notably, the detection of PTEN was significantly reduced in the treated patients, thereby warranting in-depth investigation, given the limited sample size examined in the present study.

Decreased growth differentiation factor 9, bone morphogenetic protein 15, and forkhead box O3a expressions in the ovary via ulipristal acetate.

OBJECTIVE: Folliculogenesis is a complex process involving various ovarian paracrine factors. During folliculogenesis, vitamin D3 and progesterone are significant for the proper development of follicles. This study aimed to investigate the effects of vitamin D3 and selective progesterone receptor modulator ulipristal acetate on ovarian paracrine factors. METHODS: In the study, 18 female Wistar-albino rats were randomly divided into three groups: control group (saline administration, n=6), vitamin D3 group (300 ng/day vitamin D3 oral administration, n=6), and UPA group (3 mg/kg/day ulipristal acetate oral administration, n=6). Ovarian tissue was analyzed by histochemistry and immunohistochemistry. For quantification of immunohistochemistry, the mean intensities of growth differentiation factor 9, bone morphogenetic protein 15, and forkhead box O3a expressions were measured by Image J and MATLAB. Blood samples were collected for the analysis of serum anti-Müllerian hormone levels by ELISA. RESULTS: Atretic follicles and hemorrhagic cystic structures were observed in the UPA group. After immunohistochemistry via folliculogenesis assessment markers, growth differentiation factor 9, bone morphogenetic protein 15, and cytoplasmic forkhead box O3a expressions decreased in the UPA group (p<0.05). Anti-Müllerian hormone level did not differ significantly between the experimental groups (p>0.05). CONCLUSION: Ulipristal acetate negatively affects folliculogenesis via ovarian paracrine factors. The recommended dietary vitamin D3 supplementation in healthy cases did not cause a significant change.

CDK10, CDK11, FOXO1, and FOXO3 Gene Expression in Alzheimer's Disease Encephalic Samples.

Alzheimer's disease (AD) is a progressive neuroinflammatory and neurodegenerative disorder that affects different regions of the brain. Its pathophysiology includes the accumulation of ß-amyloid protein, formation of neurofibrillary tangles, and inflammatory processes. Genetic factors are involved in the onset of AD, but they are not fully elucidated. Identification of gene expression in encephalic tissues of patients with AD may help elucidate its development. Our objectives were to characterize and compare the gene expression of CDK10, CDK11, FOXO1, and FOXO3 in encephalic tissue samples from AD patients and elderly controls, from the auditory cortex and cerebellum. RT-qPCR was used on samples from 82 individuals (45 with AD and 37 controls). We observed a statistically significant increase in CDK10 (p = 0.029*) and CDK11 (p = 0.048*) gene expression in the AD group compared to the control, which was most evident in the cerebellum. Furthermore, the Spearman test demonstrated the presence of a positive correlation of gene expression both in the auditory cortex in the AD group (r = 0.046/p = 0.004) and control group (r = 0.454/p = 0.005); and in the cerebellum in the AD group (r = 0.654 /p < 0.001). There was no statistically significant difference and correlation in the gene expression of FOXO1 and FOXO3 in the AD group and the control. In conclusion, CDK10 and CDK11 have high expression in AD patients compared to control, and they present a positive correlation of gene expression in the analyzed groups and tissues, which suggests that they play an important role in the pathogenesis of AD.

FoxO3 and oxidative stress: a multifaceted role in cellular adaptation.

Oxidative stress is a major cause of morbidity and mortality in human health and disease. In this review, we focus on the Forkhead Box (Fox) subclass O3 (FoxO3), an extensively studied transcription factor that plays a pleiotropic role in a wide range of physiological and pathological processes by regulating multiple gene regulatory networks involved in the modulation of numerous aspects of cellular metabolism, including fuel metabolism, cell death, and stress resistance. This review will also focus on regulatory mechanisms of FoxO3 expression and activity, such as crucial post-translational modifications and non-coding RNAs. Moreover, this work discusses and evidences some pathways to how this transcription factor and reactive oxygen species regulate each other, which may lead to the pathogenesis of various types of diseases. Therefore, in addition to being a promising therapeutic target, the FoxO3-regulated signaling pathways can also be used as reliable diagnostic and prognostic biomarkers and indicators for drug responsiveness.

Association of FOXO3 polymorphism (rs3800231) and clinical subphenotypes of beta thalassemic individuals

Abstract Introduction Studies have shown that the loss of the FOXO3 transcriptional function is involved in the pathophysiology of some chronic erythroid disorders, including beta-thalassemia (β-thal). Therefore, the single nucleotide polymorphism (SNP) rs3800231 (35-2764A > G) could contribute to alterations in its transcriptional activity, acting as a modifier of β-thal phenotypic manifestations. Objective and method In order to better understand the genotypic and/or allelic distributions among β-thal patients, we evaluated 83 β-thal heterozygous and 20 homozygous, compared to 117 individuals without hemoglobinopathies (control group). Additionally, we verified any influence of the FOXO3 polymorphism on clinical manifestations among β-thal homozygotes. Results We obtained higher frequencies of the wild-type homozygous (AA) and the wild-type allele (A) in the β-thal group (p< 0.0001 and p= 0.00014, respectively). The most common clinical manifestations found among β-thal homozygotes were iron overload (90%), splenomegaly (65%) and bone complications (35%), e.g., osteopenia/osteoporosis. We observed that close to 80% of the patients presenting such manifestations had the genotype AA. However, we did not find any significant involvement of the FOXO3 polymorphism in clinical manifestation occurrences. Conclusion Thus, we concluded that the SNP rs3800231 did not play a significant role as a modifier of the clinical manifestations observed in the β-thal homozygotes studied.

Involvement of Phosphorylated Akt and FOXO3a in the Effects of Growth and Differentiation Factor-9 (GDF-9) on Inhibition of Follicular Apoptosis and Induction of Granulosa Cell Proliferation After In Vitro Culture of Sheep Ovarian Tissue.

This study aimed to evaluate the effects of growth and differentiation factor-9 (GDF-9) on the morphology, activation, apoptosis, and granulosa cell proliferation of ovine preantral follicles cultured within ovarian tissue slices and to verify whether GDF-9 could influence follicular activation through the phosphatidylinositol 3-kinase/protein kinase B/forkhead box O3a (PI3K/Akt/FOXO3a) pathway. Ovine ovarian fragments were cultured in α-MEM+ or α-MEM+ with GDF-9 (1, 50, 100, 200, or 400 ng/ml) for 7 days. Apoptosis and cell proliferation were analyzed. Next, the activation of the PI3K was inhibited with LY294002, and immunostaining for p-Akt and p-FOXO3a proteins was assessed. The concentration of 50 ng/ml GDF-9 had (P < 0.05) more morphologically normal follicles compared to all treatments, except 1 ng/ml GDF-9. Moreover, 50 ng/ml GDF-9 increased primordial follicle activation compared to all treatments, except α-MEM+ and 1 ng/ml GDF-9. However, the concentration of 50 ng/ml GDF-9 showed higher cell proliferation and lower apoptosis than α-MEM+ and 1 ng/ml GDF-9 treatments. Culture of the ovarian tissue with LY294002 inhibited the activation of primordial follicles and reduced p-Akt immunostaining in both α-MEM+ and 50 ng/ml GDF-9 treatments. In addition, after culture with LY294002, the percentage of oocytes with nuclear p-FOXO3 was higher in 50 ng/ml GDF-9 than in the control medium (α-MEM+). In conclusion, after culture of ovine ovarian cortical slices, the addition of 50 ng/ml GDF-9 reduces follicular apoptosis and promotes granulosa cell proliferation likely through the involvement of phosphorylated Akt and FOXO3a.

Involvement of PTEN and FOXO3a Proteins in the Protective Activity of Protocatechuic Acid Against Cisplatin-Induced Ovarian Toxicity in Mice.

The present study evaluated the effects of protocatechuic acid (PCA) after cisplatin-induced ovarian toxicity in mice and if PTEN and FOXO3a proteins are involved in PCA action. The mice were divided into five experimental groups (five animals per group) and treated once a day for 3 days as follows: (1) the control group was pretreated with oral administration (o.p.) of saline solution, followed by an intraperitoneal (i.p.) injection of saline solution. The other groups were pretreated (o.p.) with (2) saline solution (cisplatin group), (3) N-acetylcysteine (150 mg/kg of body weight), or with (4) 20 or (5) 50 mg/kg body weight of PCA, followed by 5 mg/kg body weight (i.p.) of cisplatin. Next, the ovaries were destined to histological (morphology and activation), immunohistochemical (PCNA and cleaved caspase-3 expression), and fluorescence (reactive oxygen species [ROS], glutathione [GSH], and active mitochondria levels) analyses. Moreover, the immunoreactivity for p-PTEN and p-FOXO3a was evaluated to investigate a potential mechanism by which PCA could prevent the cisplatin-induced ovarian damage. Pretreatment with N-acetylcysteine or 20 mg/kg PCA before cisplatin preserved the percentage of normal follicles and cell proliferation as observed in the control, reduced apoptosis and ROS levels, and showed higher active mitochondria and GSH levels than the cisplatin treatment (P < 0.05). Moreover, pretreatment with 20 mg/kg PCA decreased cisplatin-induced p-PTEN and increased (P < 0.05) nuclear export of p-FOXO3a. In conclusion, PCA at 20 mg/kg reduced apoptosis, maintained cell proliferation and mitochondrial function, reduced ROS production, and increased GSH expression likely through the involvement of PTEN and FOXO3a proteins.

Angiotensin-(1-7) prevents T3-induced cardiomyocyte hypertrophy by upregulating FOXO3/SOD1/catalase and downregulating NF-ĸB.

Clinical studies have shown a correlation between thyroid disorders and cardiac diseases. High levels of triiodothyronine (T3) induce cardiac hypertrophy, a risk factor for cardiac complications and heart failure. Previous results have demonstrated that angiotensin-(1-7) is able to block T3-induced cardiac hypertrophy; however, the molecular mechanisms involved in this event have not been fully elucidated. Here, we evidenced the contribution of FOXO3 signaling to angiotensin-(1-7) effects. Angiotensin-(1-7) treatment increased nuclear FOXO3 levels and reduced p-FOXO3 levels (inactive form) in isolated cardiomyocytes. Knockdown of FOXO3 by RNA silencing abrogated the antihypertrophic effect of angiotensin-(1-7). Increased expression of antioxidant enzymes superoxide dismutase 1 (SOD1 and catalase) and lower levels of reactive oxygen species and nuclear factor-κB (NF-κB) were observed after angiotensin-(1-7) treatment in vitro. Consistent with these results, transgenic rats overexpressing angiotensin-(1-7) displayed increased nuclear FOXO3 and SOD1 levels and reduced NF-κB levels in the heart. These results provide a new molecular mechanism responsible for the antihypertrophic effect of angiotensin-(1-7), which may contribute to future therapeutic targets.

Rutin prevents cisplatin-induced ovarian damage via antioxidant activity and regulation of PTEN and FOXO3a phosphorylation in mouse model.

The aims of the present study were to evaluate the protective effects of rutin during cisplatin-induced ovarian toxicity in mice and to verify the possible involvement of the phosphatase and tension homolog (PTEN)/Forkhead box O3a (FOXO3a) pathway in the rutin actions. Mice received saline solution (control, 0.15 M, i.p.) or cisplatin (5 mg/Kg body weight, i.p.) or they were pretreated with N-acetylcysteine (positive control; 150 mg/Kg of body weight [p.o.]) or with rutin (10, 30 or 50 mg/Kg body weight, p.o.) before cisplatin (5 mg/Kg body weight, i.p.) once daily for 3 days. Next, the ovaries were harvested and destined to histological (follicular morphology and activation), immunohistochemical (cell proliferation and apoptosis) and fluorescence (reactive oxygen species [ROS], glutathione [GSH] and mitochondrial activity) analyses. Moreover, the expression of phosphorylated PTEN (p-PTEN) and FOXO3a (p-FOXO3a) were evaluated to investigate a molecular mechanism by which rutin would prevent the cisplatin-induced ovarian damage. The results showed that pretreatment with N-acetylcysteine or 10 mg/Kg rutin before cisplatin preserved the percentage of normal follicles and cell proliferation, reduced apoptosis and ROS levels and increased active mitochondria and GSH levels compared to the cisplatin treatment (P < 0.05). Cisplatin treatment increased p-PTEN and decreased p-FOXO3a expression in follicles, which was prevented by 10 mg/kg rutin. In conclusion, treatment with 10 mg/Kg rutin has the potential to protect the ovarian follicles against cisplatin-induced toxicity through its antioxidant effects and PTEN/FOXO3a pathway.

Botryosphaeran, a (1 → 3)(1 → 6)-ß-D-glucan, reduces tumor development and cachexia syndrome in obese male rats by increasing insulin sensitivity and FOXO3a activity.

Obesity is an important risk factor in tumor development. Botryosphaeran, a (1 â†’ 3)(1 â†’ 6)-ß-D-glucan, produced by the fungus Botryosphaeria rhodina (MAMB-05), is a high molecular mass, water-soluble exopolysaccharide. It consists of a main chain of (1 â†’ 3)-linked ß-d-glucose units, with a degree of branching of ~22% at carbon-6 with glucose and gentiobiose residues linked through ß-(1 â†’ 6)-bonds, and presents a triple helix conformation. Botryosphaeran presents anticlastogenic, antiproliferative, pro-apoptotic and anti-obesogenic activities. This study evaluated the effects of botryosphaeran on tumor development in obesity and analyzed its mechanism of action. Obesity was induced in male Wistar rats by a high-fat/high-sugar diet. After 9 weeks, rats were divided into two groups: Obese Tumor (OT) and Obese Tumor Botryosphaeran (OTB), and inoculated with 1 × 107 Walker-256 tumor cells, and treatment with botryosphaeran (30 mg/kg b.w./day via gavage for 15 days) commenced. On the 11th week, biological parameters, tumor development, metabolic profile, erythrogram and protein expression were evaluated. Botryosphaeran significantly reduced tumor growth, body-weight loss and cachexia. Furthermore, botryosphaeran decreased mesenteric fat and insulin resistance, corrected macrocytic anemia, and increased Forkhead transcription factor-3a (FOXO3a) activity. Our study demonstrated the potential role of botryosphaeran in the management of cancer in tumor-bearing obese rats by increasing insulin sensitivity and FOXO3a activity.

cAMP-dependent protein kinase inhibits FoxO activity and regulates skeletal muscle plasticity in mice.

Although we have shown that catecholamines suppress the activity of the Ubiquitin-Proteasome System (UPS) and atrophy-related genes expression through a cAMP-dependent manner in skeletal muscle from rodents, the underlying mechanisms remain unclear. Here, we report that a single injection of norepinephrine (NE; 1 mg kg-1 ; s.c) attenuated the fasting-induced up-regulation of FoxO-target genes in tibialis anterior (TA) muscles by the stimulation of PKA/CREB and Akt/FoxO1 signaling pathways. In addition, muscle-specific activation of PKA by the overexpression of PKA catalytic subunit (PKAcat) suppressed FoxO reporter activity induced by (1) a wild-type; (2) a non-phosphorylatable; (3) a non-phosphorylatable and non-acetylatable forms of FoxO1 and FoxO3; (4) downregulation of FoxO protein content, and probably by (5) PGC-1α up-regulation. Consistently, the overexpression of the PKAcat inhibitor (PKI) up-regulated FoxO activity and the content of Atrogin-1 and MuRF1, as well as induced muscle fiber atrophy, the latter effect being prevented by the overexpression of a dominant negative (d. n.) form of FoxO (d.n.FoxO). The sustained overexpression of PKAcat induced fiber-type transition toward a smaller, slower, and more oxidative phenotype and improved muscle resistance to fatigue. Taken together, our data provide the first evidence that endogenous PKA activity is required to restrain the basal activity of FoxO and physiologically important to maintain skeletal muscle mass.

PTEN and FOXO3 expression in the prenatal and postnatal human ovary.

PURPOSE: The objective of this study was to analyse the expression and cellular localization of FOXO3, pFOXO3 and PTEN throughout human ovary development both before and after birth. METHODS: Foetal, pubertal and adult paraffin-embedded ovarian samples were analysed by immunohistochemistry for cellular localization of FOXO3, pFOXO3 and PTEN proteins. Protein and mRNA expression were analysed by western blot and real time PCR, respectively, from fresh biopsies. RESULTS: PTEN was not detected by immunohistochemistry in germ cells and follicles of foetal, pubertal and adult ovaries. Occasional PTEN immunoreactive granulosa cells were found in atretic antral follicles in the adult ovary. Western blot analysis showed low levels of PTEN protein. Nuclear FOXO3-expressing primordial follicles represented a variable proportion of the ovarian reserve. The presence of FOXO3-expressing primordial follicles was very low in foetal ovary; although always represented in a low proportion, prevalence increased during pubertal and adult life. CONCLUSION: Our results seem to indicate that two subpopulations of primordial follicles, i.e. nuclear FOXO3-expressing and no FOXO3-expressing primordial follicles are found in the postnatal human ovary. This scenario suggests that FOXO3 could be acting as in the mouse model, preventing primordial follicle activation. However, the strategy would not be an "all or nothing" system as in mouse ovary but rather a selected subpopulation of primordial follicles preserved to ensure long-term fertility.

Airborne Particulate Matter (PM10) Inhibits Apoptosis through PI3K/AKT/FoxO3a Pathway in Lung Epithelial Cells: The Role of a Second Oxidant Stimulus.

Outdoor particulate matter (PM10) exposure is carcinogenic to humans. The cellular mechanism by which PM10 is associated specifically with lung cancer includes oxidative stress and damage to proteins, lipids, and DNA in the absence of apoptosis, suggesting that PM10 induces cellular survival. We aimed to evaluate the PI3K/AKT/FoxO3a pathway as a mechanism of cell survival in lung epithelial A549 cells exposed to PM10 that were subsequently challenged with hydrogen peroxide (H2O2). Our results showed that pre-exposure to PM10 followed by H2O2, as a second oxidant stimulus increased the phosphorylation rate of pAKTSer473, pAKTThr308, and pFoxO3aSer253 2.5-fold, 1.8-fold, and 1.2-fold, respectively. Levels of catalase and p27kip1, which are targets of the PIK3/AKT/FoxO3a pathway, decreased 38.1% and 62.7%, respectively. None of these changes had an influence on apoptosis; however, the inhibition of PI3K using the LY294002 compound revealed that the PI3K/AKT/FoxO3a pathway was involved in apoptosis evasion. We conclude that nontoxic PM10 exposure predisposes lung epithelial cell cultures to evade apoptosis through the PI3K/AKT/FoxO3a pathway when cells are treated with a second oxidant stimulus.

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.

Inhibitory effect of Astragalus polysaccharide on osteoporosis in ovariectomized rats by regulating FoxO3a /Wnt signaling pathway.

PURPOSE: To investigate inhibitory effect of Astragalus polysaccharide (APS) on osteoporosis in ovariectomized rats by regulating FoxO3a/Wnt2 signaling pathway. METHODS: Postmenopausal osteoporosis (PMOP) animal model was developed by excising the bilateral ovaries of rats. The model rats were administered with APS (200 mg/kg, 400 mg/kg, 800 mg/kg) by intragastric administration once daily for 12 weeks. Bone density, bone metabolism index and oxidative stress index were measured in all groups. Furthermore, the regulation of APS of FoxO3a / Wnt2 signaling pathway was observed. RESULTS: APS has an estrogen-like effect, which can increase bone mass, lower serum ALP and BGP values, increase blood calcium content, and increase bone density of the femur and vertebrae in rats. At the same time, APS can increase the bone mineral content of the femur, increase the maximum stress, maximum load and elastic modulus of the ovariectomized rats, improve oxidative stress in rats by increasing the gene expression of ß-catenin and Wnt2 mRNA and inhibiting the gene expression of FoxO3a mRNA. CONCLUSION: Astragalus polysaccharide can effectively alleviate oxidative stress-mediated osteoporosis in ovariectomized rats, which may be related to its regulation of FoxO3a/Wnt2/ß-catenin pathway.

Exome Sequencing Reveals the POLR3H Gene as a Novel Cause of Primary Ovarian Insufficiency.

CONTEXT: Primary ovarian insufficiency (POI) is a cause of female infertility. However, the genetic etiology of this disorder remains unknown in most patients with POI. OBJECTIVE: To investigate the genetic etiology of idiopathic POI. PATIENTS AND METHODS: We performed whole-exome sequencing of 11 families with idiopathic POI. To gain insights into the potential mechanisms associated with this mutation, we generated two mouse lines via clustered regularly interspaced short palindromic repeats/Cas9 technology. RESULTS: A pathogenic homozygous missense mutation (c.149A>G; p.Asp50Gly) in the POLR3H gene in two unrelated families was identified. Pathogenic mutations in this subunit have not been associated with human disorders. Loss-of-function Polr3h mutation in mice caused early embryonic lethality. Mice with homozygous point mutation (Polr3hD50G) were viable but showed delayed pubertal development, characterized by late first estrus or preputial separation. The Polr3hD50G female and male mice showed decreased fertility later in life, associated with small litter size and increased time to pregnancy or to impregnate a female. Polr3hD50G mice displayed decreased expression of ovarian Foxo3a and lower numbers of primary follicles. CONCLUSION: Our manuscript provides a case of POI caused by missense mutation in POLR3H, expanding the knowledge of molecular pathways of the ovarian function and human infertility. Screening of the POLR3H gene may elucidate POI cases without previously identified genetic causes, supporting approaches of genetic counseling.

Omega-3 multiple effects increasing glucocorticoid-induced muscle atrophy: autophagic, AMPK and UPS mechanisms.

Muscle atrophy occurs in many conditions, including use of glucocorticoids. N-3 (omega-3) is widely consumed due its healthy properties; however, concomitant use with glucocorticoids can increase its side effects. We evaluated the influences of N-3 on glucocorticoid atrophy considering IGF-1, Myostatin, MEK/ERK, AMPK pathways besides the ubiquitin-proteasome system (UPS) and autophagic/lysosomal systems. Sixty animals constituted six groups: CT, N-3 (EPA 100 mg/kg/day for 40 days), DEXA 1.25 (DEXA 1.25 mg/kg/day for 10 days), DEXA 1.25 + N3 (EPA for 40 days + DEXA 1.25 mg/kg/day for the last 10 days), DEXA 2.5 (DEXA 2.5 mg/kg/day for 10 days), and DEXA 2.5 + N3 (EPA for 40 days + DEXA 2.5 mg/kg/day for 10 days). Results: N-3 associated with DEXA increases atrophy (fibers 1 and 2A), FOXO3a, P-SMAD2/3, Atrogin-1/MAFbx (mRNA) expression, and autophagic protein markers (LC3II, LC3II/LC3I, LAMP-1 and acid phosphatase). Additionally, N-3 supplementation alone decreased P-FOXO3a, PGC1-alpha, and type 1 muscle fiber area. Conclusion: N-3 supplementation increases muscle atrophy caused by DEXA in an autophagic, AMPK and UPS process.

Inhibitory effect of Astragalus polysaccharide on osteoporosis in ovariectomized rats by regulating FoxO3a /Wnt signaling pathway

Abstract Purpose: To investigate inhibitory effect of Astragalus polysaccharide (APS) on osteoporosis in ovariectomized rats by regulating FoxO3a/Wnt2 signaling pathway. Methods: Postmenopausal osteoporosis (PMOP) animal model was developed by excising the bilateral ovaries of rats. The model rats were administered with APS (200 mg/kg, 400 mg/kg, 800 mg/kg) by intragastric administration once daily for 12 weeks. Bone density, bone metabolism index and oxidative stress index were measured in all groups. Furthermore, the regulation of APS of FoxO3a / Wnt2 signaling pathway was observed. Results: APS has an estrogen-like effect, which can increase bone mass, lower serum ALP and BGP values, increase blood calcium content, and increase bone density of the femur and vertebrae in rats. At the same time, APS can increase the bone mineral content of the femur, increase the maximum stress, maximum load and elastic modulus of the ovariectomized rats, improve oxidative stress in rats by increasing the gene expression of β-catenin and Wnt2 mRNA and inhibiting the gene expression of FoxO3a mRNA. Conclusion: Astragalus polysaccharide can effectively alleviate oxidative stress-mediated osteoporosis in ovariectomized rats, which may be related to its regulation of FoxO3a/Wnt2/β-catenin pathway.

Metabolic gene expression and epigenetic effects of the ketone body ß-hydroxybutyrate on H3K9ac in bovine cells, oocytes and embryos.

The rapid decline in fertility that has been occurring to high-producing dairy cows in the past 50 years seems to be associated with metabolic disturbances such as ketosis, supporting the need for research to improve our understanding of the relations among the diet, metabolism and embryonic development. Recently, the ketone body ß-hydroxybutyrate (BOHB) was demonstrated to be a potent inhibitor of histone deacetylases (HDACs). Herein, we performed a series of experiments aiming to investigate the epigenetic effects of BOHB on histone acetylation in somatic cells, cumulus-oocyte complexes (COCs) and somatic cell nuclear transfer (SCNT) embryos. Treatment with BOHB does not increase histone acetylation in cells but stimulates genes associated with ketolysis and master regulators of metabolism. We further demonstrated that maturing COCs with high levels of BOHB does not affect their maturation rate or histone acetylation but increases the expression of PPARA in cumulus cells. Treatment of somatic cell nuclear transfer zygotes with BOHB causes hyperacetylation, which is maintained until the blastocyst stage, causing enhanced FOXO3A expression and blastocyst production. Our data shed light on the epigenetic mechanisms caused by BOHB in bovine cells and embryos and provide a better understanding of the connection between nutrition and reproduction.