Social defeat stress impairs systemic iron metabolism by activating the hepcidin-ferroportin axis.

Chronic psychological stress has been reported to decrease circulating iron concentrations and impair hematopoiesis. However, the underlying mechanisms remain unclear. This study aimed to investigate the effects of psychological stress on biological iron metabolism by using the social defeat stress (SDS) model, a widely used model of depression. Compared with control mice, mice subjected to SDS (SDS mice) had lower social interaction (SI) behavior. The SDS mice also showed impaired hematopoiesis, as evidenced by reduced circulating red blood cell counts, elevated reticulocyte counts, and decreased plasma iron levels. In the SDS mice, the iron contents in the bone marrow decreased, whereas those in the spleen increased, suggesting dysregulation in systemic iron metabolism. The concentrations of plasma hepcidin, an important regulator of systemic iron homeostasis, increased in the SDS mice. Meanwhile, the concentrations of ferroportin, an iron transport protein negatively regulated by hepcidin, were lower in the spleen and duodenum of the SDS mice than in those of the control mice. Treatment with dalteparin, a hepcidin inhibitor, prevented the decrease in plasma iron levels in the SDS mice. The gene expression and enzyme activity of furin, which converts the precursor hepcidin to active hepcidin, were high and positively correlated with plasma hepcidin concentration. Thus, furin activation might be responsible for the increased plasma hepcidin concentration. This study is the first to show that psychological stress disrupts systemic iron homeostasis by activating the hepcidin-ferroportin axis. Consideration of psychological stressors might be beneficial in the treatment of diseases with iron-refractory anemia.

Effects of Importin α1/KPNA1 deletion and adolescent social isolation stress on psychiatric disorder-associated behaviors in mice.

Importin α1/KPNA1 is a member of the Importin α family widely present in the mammalian brain and has been characterized as a regulator of neuronal differentiation, synaptic functionality, and anxiety-like behavior. In humans, a de novo mutation of the KPNA1 (human Importin α5) gene has been linked with schizophrenia; however, the precise roles of KPNA1 in disorder-related behaviors are still unknown. Moreover, as recent studies have highlighted the importance of gene-environment interactions in the development of psychiatric disorders, we investigated the effects of Kpna1 deletion and social isolation stress, a paradigm that models social stress factors found in human patients, on psychiatric disorder-related behaviors in mice. Through assessment in a behavioral battery, we found that Kpna1 knockout resulted in the following behavioral phenotype: (1) decreased anxiety-like behavior in an elevated plus maze test, (2) short term memory deficits in novel object recognition test (3) impaired sensorimotor gating in a prepulse inhibition test. Importantly, exposure to social isolation stress resulted in additional behavioral abnormalities where isolated Kpna1 knockout mice exhibited: (1) impaired aversive learning and/or memory in the inhibitory avoidance test, as well as (2) increased depression-like behavior in the forced swim test. Furthermore, we investigated whether mice showed alterations in plasma levels of stress-associated signal molecules (corticosterone, cytokines, hormones, receptors), and found that Kpna1 knockout significantly altered levels of corticosterone and LIX (CXCL5). Moreover, significant decreases in the level of prolactin were found in all groups except for group-housed wild type mice. Our findings demonstrate that Kpna1 deletion can trigger widespread behavioral abnormalities associated with psychiatric disorders, some of which were further exacerbated by exposure to adolescent social isolation. The use of Kpna1 knockout mice as a model for psychiatric disorders may show promise for further investigation of gene-environment interactions involved in the pathogenesis of psychiatric disorders.

Decreased Colonic Guanylin/Uroguanylin Expression and Dried Stool Property in Mice With Social Defeat Stress.

Psychological stress is deeply involved in the pathophysiology of not only mental illness but also functional gastrointestinal disorders. In the present study, we examined the relationship between psychological stress and abnormality of stool properties, focusing on the alteration of plasma glucocorticoid and guanylin (GN)/uroguanylin (UGN) expression in the colon. A murine model of chronic social defeat stress (CSDS) was established by exposing a C57BL/6N intruder mouse to a CD-1 aggressor mouse for 3-5 min. Thereafter the mice were kept in the same cage but separated by a divider for the remainder of the day. This procedure was repeated for 10 consecutive days, and then a social interaction test was performed to evaluate social avoidance. Fresh fecal and blood samples were collected for stool property analysis and measurement of the plasma glucocorticoid level by ELISA. The expression of GN, UGN, and guanylate cyclase 2C in the colonic tissues was examined by real-time RT-PCR and immunohistochemistry. Moreover, Lovo cells were stimulated with dexamethasone, and the expression of GN/UGN mRNA was examined. In the CSDS group, the time spent in the social interaction zone was significantly shorter when the CD-1 aggressor mouse was present than when it was absent. The social interaction ratio was also significantly lower in the CSDS group relative to the controls. The mean Bristol scale score was significantly lower in the CSDS group, but the fecal sodium concentration did not differ between CSDS mice and controls. The level of plasma corticosterone was significantly higher in the CSDS group than in the controls immediately after the 10th day of CSDS. The expression of both GN and UGN was significantly decreased in the CSDS mice. GN was expressed in all colonic epithelial cells, and UGN was expressed in ovoid or pyramidal epithelial cells in the colonic mucosa. The expression of both GN and UGN was significantly decreased in the CSDS mice relative to controls. The expression of both GN and UGN was significantly suppressed in Lovo cells upon stimulation with dexamethasone. Psychological stress-induced glucocorticoid may suppress colonic GN/UGN expression, resulting in a change in stool properties leading to constipation.

The Clock Genes Are Involved in The Deterioration of Atopic Dermatitis after Day-and-Night Reversed Physical Stress in NC/Nga Mice.

BACKGROUND: In modern society, irregular lifestyles are a problem. It is well known that Atopic Dermatitis (AD) occurs during physical stress in people with an irregular lifestyle. We evaluated the influence that day-and-night reversal physical stress has on AD. METHODS: Six-week-old specific-pathogen-free and conventional NC/Nga male mice were used. For the day-and-night reversal procedure, the mice ran on a treadmill at a slow speed of 10 m/min for 12 h (between 8:00 and 20:00). Then, between 20:00 and 8:00, we put the mice in a dark place. This treatment was repeated every day for two weeks. The behavioral circadian rhythm of the mice was evaluated with the open field test. Then, the mice were sacrificed and histological examinations of the tissues, the expression of peptide hormones, corticosterone, Immunoglobulin E, histamine, and cytokines was performed using an enzyme-linked immunosorbent assay. RESULTS: In the treadmill-treated conventional NC/Nga mice, AD symptoms were deteriorated compared with the non-treated conventional NC/Nga mice. The levels of Period (Per) 2, Clock, and brain and muscle arnt-like protein 1 (Bmal1) in the skin were increased constantly in the treadmill-treated conventional mice. Furthermore, the expression of Retinoic Acid-related Orphan Receptor (ROR)α, which activates Bmal1, was increased in the treadmill-treated conventional mice compared with the non-treated conventional mice. In addition, when non-treated conventional mice were administrated by the agonist of RORα, AD symptoms were deteriorated similar to treadmill-treated conventional mice. CONCLUSION: In the day-and-night reversal mice, the clock genes were increased constantly, indicating that this is a factor that deteriorated AD.

An Inhibitor of Casein Kinase 1ε/δ (PF670462) Prevents the Deterioration of Dextran Sodium Sulfate-induced Ulcerative Colitis Caused by UVB Eye Irradiation.

Although we previously reported the exacerbation of dextran sodium sulfate (DSS)-induced ulcerative colitis by ultraviolet (UV) B eye irradiation, we do not yet understand the mechanism behind this phenomenon. In this study, we examined the relationship between the deterioration of DSS-induced ulcerative colitis and clock genes. We induced a mouse model of ulcerative colitis by administering DSS for 5 days, and administered UVB eye irradiation on each day of the DSS treatment period. The DSS-induced ulcerative colitis was deteriorated by the UVB eye irradiation. The levels of Clock, brain and muscle arnt-like protein 1 (Bmal1), reverse orientation c-erb A gene (Rev-Erb)α, RAR-related orphan receptor gamma (RORγt), and interleukin (IL)-17 in the colon were increased by UVB eye irradiation in the DSS-treated mice (UVB/DSS-treated mice). Conversely, the nuclear factor, interleukin 3 regulated (NFIL-3) levels in the colon were lower after UVB eye irradiation. The Casein Kinase 1ε/δ inhibitor (PF670462) administration, which is a Clock/Bmal1 inhibitor (PER2 activator), inhibited the deterioration caused by UVB eye irradiation. These results suggest that the UVB eye irradiation-mediated exacerbation of DSS-induced ulcerative colitis depends on IL-17 produced in response to alterations in clock genes.

Long-term UVA eye irradiation causes decreased learning ability in mice.

BACKGROUND: Long-term eye radiation with ultraviolet A (UVA) denatures the cells of the cerebral hippocampus. However, the influence on memory and learning ability in mice is not known. METHODS: HR-1 mice were used. We irradiated the eyes or dorsal skin of the mice with UVA at a dose of 110 kJ/m(2) using an FL20SBLB-A lamp for 6, 12, and 24 months. RESULTS: The mean escape latency in the water maze was significantly increased in the UVA-irradiated mice in comparison with that seen in the controls. In the mice in which UVA eye irradiation was performed for 24 months, the depression of memory and learning ability was remarkable. The acetylcholinesterase activity, choline acetyltransferase activity, and acetylcholine content in the brain in the UVA eye-irradiated mice were significantly less than those observed in the control mice. Furthermore, during UVA eye irradiation, the levels of ß-amyloid (Aß), γ-secretase, which produces Aß peptide, and advanced glycation end products increased. Moreover, the effects of UVA eye irradiation increased with the duration of irradiation (or aging), and the introduction of glucose into the brain also decreased with UVA eye irradiation. CONCLUSIONS: These results indicate that UVA eye irradiation induces a decreased memory and learning ability in mice.

Stress-Induced Glucocorticoid Release Upregulates Uncoupling Protein-2 Expression and Enhances Resistance to Endotoxin-Induced Lethality.

OBJECTIVE: Although psychological and/or physiological stress has been well documented to influence immune responses, the precise mechanism for immunomodulation remains to be elucidated. The present work describes the role of the hypothalamic-pituitary-adrenal (HPA) axis in the mechanism of stress-mediated enhanced-resistance to lethality after lipopolysaccharide (LPS) injection. METHODS/RESULTS: Preconditioning with restraint stress (RS) resulted in enhanced activation of the HPA axis in response to LPS injection and suppressed LPS-induced release of proinflammatory cytokines and nitric oxide metabolites. Melanocortin 2 receptor-deficient mice (MC2R(-/-)) failed to increase plasma levels of glucocorticoids in response to LPS injection, and exhibited high sensitivity to LPS-induced lethality with enhanced release of proinflammatory cytokines as compared with MC2R(+/-) mice. Real-time PCR analysis revealed that RS induced upregulation of uncoupling protein-2 (UCP2) in macrophages in the lung and the liver of MC2R(+/-), but not of MC2R(-/-), mice. In addition, RS increased UCP2-dependent uncoupling activity of isolated mitochondria from the liver of MC2R(+/-), but not of MC2R(-/-), mice. In vitro study revealed that corticosterone and dexamethasone directly increased UCP2 expression in mouse RAW 264.7 macrophages and suppressed the generation of LPS-induced mitochondrial reactive oxygen species (ROS) and TNF-α production. Knockdown of UCP2 by small interfering RNA blunted the dexamethasone action for suppressing LPS-induced mitochondrial ROS and TNF-α production. CONCLUSION: The present work suggests that RS enhances activation of the HPA axis to release glucocorticoids and upregulation of UCP2 in macrophages, thereby increasing the resistance to endotoxin-induced systemic inflammation and death.

Anti-inflammatory effect of water-soluble complex of 1'-acetoxychavicol acetate with highly branched ß-1,3-glucan on contact dermatitis.

The anti-inflammatory effect on contact dermatitis of the water solubilized 1'-Acetoxychavicol Acetate (ACA) by complexation with ß-1,3-glucan isolated form Aureobasidium pullulans black yeast is reported. It is well-known that ACA possesses a function to inhibit the activation of NF-κB by which genes encoding proinflammatory cytokines, chemokines, and growth factors are regulated. However, because ACA is quite insoluble in water, its usefulness has been extremely limited. On the other hand, a triple-helical polysaccharide ß-1,3-glucan can include hydrophobic compounds into intrastrand hydrophobic cavity and solubilize poorly water-soluble compounds. In this study, solubilization of ACA by complexation with highly branched ß-1,3-glucan was achieved. The effect of anti-inflammatory response of water-soluble ACA complex with ß-1,3-glucan was confirmed in vitro and in vivo.

Cross-talk between HPA-axis-increased glucocorticoids and mitochondrial stress determines immune responses and clinical manifestations of patients with sepsis.

Various stressors activate the hypothalamo-pituitary-adrenal axis (HPA-axis) that stimulates adrenal secretion of glucocorticoids, thereby playing critical roles in the modulation of immune responses. Transcriptional regulation of nuclear genes has been well documented to underlie the mechanism of glucocorticoid-dependent modulation of cytokine production and immune reactions. Glucocorticoids also regulate inflammatory responses via non-genomic pathways in cytoplasm and mitochondria. Recent studies have revealed that glucocorticoids modulate mitochondrial calcium homeostasis and generation of reactive oxygen species (ROS). Although redox status and ROS generation in inflammatory cells have been well documented to play important roles in defense against pathogens, the roles of glucocorticoids and mitochondria in the modulation of immunological responses remain obscure. This review describes the role of stress-induced activation of the HPA-axis and glucocorticoid secretion by the adrenal gland in mitochondria-dependent signaling pathways that modulate endotoxin-induced inflammatory reactions and innate immunity.

Role of adrenocorticotropic hormone in the modulation of pollinosis induced by pollen antigens.

BACKGROUND: A mild restraint stressor suppressed an increase in the levels of Th2-dependent cytokines and IgE, thereby reducing the symptoms of pollinosis. In the present study, to clarify the mechanism of action of adrenocorticotropic hormone (ACTH) in improving the symptoms of pollinosis, we studied the effects of ACTH on the plasma level of histamine, mast cell number in nasal-associated lymphoid tissue (NALT) and the T cell differentiation in splenocytes. METHODS: The role of ACTH in the development of pollen antigen-induced pollinosis was studied in mice. Allergic symptoms and parameters were measured on day 17 after sensitization. To investigate the effects of ACTH on T cell differentiation, we stimulated splenocytes obtained from control mice with ACTH and CD3/CD28 in vitro, and measured the cytokine production in the culture supernatant. RESULTS: The plasma levels of IL-10, IgE and histamine and mast cell number in NALT were increased in the sensitized animals in association with a concomitant increase in the incidence of sneezing and nasal rubbing. The intraperitoneal administration of ACTH decreased the IL-10, IgE and histamine levels in the plasma and mast cell number in NALT, while increasing the IFN-γ level and suppressing the incidence of nasal rubbing. Furthermore, the production of IFN-γ increased, while the IL-4 level was suppressed after 2 days in culture. CONCLUSIONS: The present findings showed that ACTH directly affects T cell differentiation and promotes Th1-type reactions. The regulation of the Th1/Th2 balance by ACTH may result in a decrease in the pathological features of pollinosis.

UVA irradiation of the eye modulates the contact hypersensitivity of the skin and intestines by affecting mast cells in mice.

BACKGROUND: Ultraviolet A (UVA) irradiation before allergic sensitization induces immunosuppression, but the precise mechanism remained unclear. In this study, we examined the influence of UVA irradiation of the eye on contact hypersensitivity (CHS) and the role of mast cells in CHS. METHODS: We used two types of haptens, fluorescein isothiocyanate (FITC: a Th2 type hapten) and 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone: a Th1 type hapten). A 300 kJ/m(2) dose of UVA irradiation was delivered to the eyes. After UVA irradiation, we sensitized abdominal shaved skin and challenged the ear epidermis and colons of these mice with each hapten. RESULTS: After UVA irradiation, the CHS of the skin and colon were not inhibited in the FITC-sensitized mice. However, in the oxazolone-sensitized mice, only the CHS of the skin was inhibited by UVA irradiation. The inflammation of the colon became more severe after UVA irradiation. In mast cell-deficient (W/Wv) mice sensitized to FITC, the CHS was weaker than that in WT mice. Moreover, the reduction of immunosuppression in ear swelling was seen for one of the two models they used. CONCLUSIONS: These results suggest that the mast cells induced by UVA irradiation of the eye have different roles in the epidermis and colon and have different responses to different haptens.

Isolation of diploid baker's yeast capable of strongly activating immune cells and analyses of the cell wall structure.

Diploid baker's yeast capable of strongly activating a mouse macrophage was constructed based on haploid mutant AQ-37 obtained previously. The obtained strain BQ-55 activated also human immune cells. To clarify a factor for the activation, the cell wall structure, especially the ß-glucan structure, was analyzed, suggesting that the length of branching, ß-1,6-glucan, may be one of the factors.

Gender difference in tumor necrosis factor-α production in human neutrophils stimulated by lipopolysaccharide and interferon-γ.

The gender difference in tumor necrosis factor-α (TNF-α) production in human neutrophils stimulated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ) was explored by using peripheral blood neutrophils from young men and women. As compared with female neutrophils, male neutrophils released greater amounts of TNF-α, and exhibited stronger activation of mitogen-activated protein kinases and phosphatidylinositol 3-kinase in response to LPS stimulation. LPS-induced TNF-α production was markedly enhanced by pretreatment of cells with IFN-γ, and IFN-γ-mediated priming in male neutrophils was significantly greater than that in female neutrophils. Male neutrophils showed higher expression of TLR4, but not IFN-γ receptors, than female neutrophils, and its expression was increased by stimulation with IFN-γ or IFN-γ plus LPS. These findings indicate that male neutrophils show higher responsiveness to stimulation with LPS and IFN-γ than female neutrophils, and suggest that the gender difference in neutrophil responsiveness to LPS and IFN-γ is partly responsible for that in the outcome of sepsis, in which premenopausal women show a favorable prognosis as compared with men.

Potent immunomodulating effects of bran extracts of traditional Japanese millets on nitric oxide and cytokine production of macrophages (RAW264.7) induced by lipopolysaccharide.

To estimate the potent immunomodulating effects of different types of traditional Japanese millet, we analyzed the effect of bran extracts of foxtail millet (Awa in Japanese), barnyard millet (Hie) and proso millet (Mochi-kibi) on nitric oxide (NO) and inflammatory cytokine production induced by lipopolysaccharide (LPS) in a mouse macrophage cell line (RAW264.7 cells). All methanol extracts of these millet brans showed suppressive activities against the production of NO and inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6 in LPS-stimulated macrophages, which were not responsible for their cytotoxic activities. These immunosuppressive activities were roughly proportional to the contents of the phenolic compounds in their extracts. Especially, the extract of proso millet exhibited the strongest immunosuppressing effect, which was associated with the highest content of phenolic compound. However, the extracts did not exhibit significant suppressive effects on the production of an anti-inflammatory cytokine, IL-10, in the same macrophage culture system. These results suggest that traditional Japanese millets have potent immunomodulating activities against the production of NO and cytokine production in activated macrophages.

A promoter variant in the chitinase 3-like 1 gene is associated with serum YKL-40 level and personality trait.

The chitinase 3-like 1 (CHI3L1) gene, a cellular survival factor against several environmental and psychosocial stresses, has been sown to be more highly expressed in the hippocampus and prefrontal cortex of patients with schizophrenia than unaffected individuals. We recently reported a significant association between schizophrenia and SNP rs4950928, which is located in the promoter region of the CHI3L1 gene, in a Japanese population. The G-allele at this SNP in the gene has been associated with higher transcriptional activity in a luciferase reporter assay and with higher mRNA levels in the peripheral blood cells of patients with schizophrenia. We investigated the impact of the CHI3L1 polymorphism rs4950928 on serum YKL-40 levels, the protein product of CHI3L1. We found that individuals with the G-allele, who were more prevalent among patients with schizophrenia, had significantly higher serum YKL-40 levels (p=0.043). Personality traits are considered to be an important aspect of schizophrenia primarily because they may influence symptoms and social functioning. Personality trait analyses using the temperament and character inventory (TCI) indicated that schizophrenic patients have a unique personality profile that appears to be present across cultures. We hypothesized that higher serum YKL-40 levels are associated with personality trait in patients with schizophrenia. Thus, we next examined the impact of the risk CHI3L1 polymorphism on personality traits using the TCI. We found that individuals with the G-allele had significantly higher self-transcendence scores (p=0.0054). These findings suggest possible associations between the SNP in the CHI3L1 gene, the risk for schizophrenia, and higher serum YKL-40 levels and personality traits in a Japanese population.

Mitochondrial density contributes to the immune response of macrophages to lipopolysaccharide via the MAPK pathway.

We investigated the role of mitochondrial reactive oxygen species (ROS) in the response of macrophages to lipopolysaccharide (LPS) using RAW 264.7 cells and their ρ(o) cells lacking mitochondria. Mitochondrial density, respiratory activity and related proteins in ρ(o) cells were significantly lower than those in RAW cells. LPS rapidly stimulated mitochondrial ROS prior to cytokine secretion, such as TNF-α and IL-6, from RAW 264.7 cells by activating the MAPK pathway, while the response was attenuated in ρ(o) cells. Exposure of ρ(o) cells to H(2)O(2) partially restored the secretion of cytokines induced by LPS. These results suggest that mitochondrial density and/or the respiratory state contribute to intracellular oxidative stress, which is responsible for the stimulation of LPS-induced MAPK signaling to enhance cytokine release from macrophages.

Mitochondria determine the efficacy of anticancer agents that interact with DNA but not the cytoskeleton.

Although chemotherapy is an important method for the treatment of patients with cancer, its efficacy is limited because of different sensitivities of tumor cells to anticancer agents and/or side effects on normal tissues. The present work demonstrates that mitochondria play a crucial role in the apoptosis of cancer cells induced by anticancer agents that interact with DNA but not with the cytoskeleton. Agents that interact with DNA selectively enhanced generation of reactive oxygen species (ROS) in mitochondria, released cytochrome c, and activated caspase-9 and caspase-3 to induce apoptosis of mesothelioma H2052 cells but not their ρ(0) cells, which lack mitochondrial DNA (mtDNA). The sensitivity of a variety of cells to the agents showed positive correlation with the amounts of their mitochondria. In contrast, agents that selectively affect the cytoskeleton activated caspase-8 and caspase-3 and equally induced apoptosis of both H2052 and their ρ(0) cells by a mitochondria-independent mechanism. The results suggest that mtDNA is a potential target for the anticancer agents that interact with DNA to induce ROS-dependent apoptosis of cancer cells, whereas agents that affect the cytoskeleton induce cell death by a mitochondria- and ROS-independent mechanism. The present observation is important for the selection of medicine for chemotherapy of patients with cancer.

Ultraviolet-A irradiation to the eye modulates intestinal mucosal functions and properties of mast cells in the mouse.

We previously reported that topical irradiation of the eye by ultraviolet-B (UVB) activated hypothalamo-pituitary-adrenal axis (HPA-A) of the mouse to increase 3, 4-dihydroxyphenylalanine (DOPA)-positive melanocytes in the skin by an inducible nitric oxide synthase (iNOS)-dependent mechanism. This work demonstrates that irradiation of the eye by ultraviolet-A (UVA) specifically increased DOPA-positive cells in the mucosa of the jejunum and colon of C57BL/6J mice by some HPA- and iNOS-independent mechanism. UVA-induced increase in DOPA-positive cells in the intestine was inhibited by the administration of hexamethonium or prazosin plus propranolol, blockers for the sympathetic nervous system. UVA irradiation of the eye increased DOPA- and histidine decarboxylase (HDC)-positive cells in the intestinal mucosa of both C57BL/6J and WBB6F1/J mice but not in the mutant strain W/Wv of the latter that lack mast cells. UVA irradiation of the eye suppressed the intestinal peristalsis of control, hypophysectomized or iNOS(-/-) C57BL/6J mice by the mechanism that was inhibited by hexamethonium or prazosin plus propranolol. These observations suggest that UVA irradiation of the eye stimulated the sympathetic nervous system to increase the mucosal DOPA- and HDC-positive mast cells and suppressed the peristalsis of the small intestine of the mouse.

Effect of oxidative stress during repeated ovulation on the structure and functions of the ovary, oocytes, and their mitochondria.

We previously reported that superoxide generated in the ovary induces apoptosis of granulosa cells to break down follicular walls, thereby supporting ovulation in rodents, and suggested that oxidative stress underlies the mechanism of ovarian aging. To test this hypothesis, we successfully induced ovulation repeatedly in mice by sequentially administrating pregnant mare serum gonadotropin, human chorionic gonadotropin, and prostaglandin F2alpha. Kinetic analysis revealed that the number of ovulated oocytes decreased significantly with repeated cycles of ovulation with a concomitant decrease in the gene expression of mitochondrial transcription factor A and nuclear respiratory factor 1 and an increase in oocytes having abnormally distributed mitochondria. Repeated ovulation decreased the amounts of mitochondrial DNA and increased 8-hydroxydeoxyguanosine in oocytes. Cell culture analysis of the in vivo fertilized oocytes revealed that their maturation from two cells to blastocyst was inhibited significantly by repeated ovulation. All these events induced by repeated ovulation were suppressed by oral administration of L-carnitine. These results suggest that oxidative stress associated with ovulation underlies the mechanism of ovarian aging and that L-carnitine may have therapeutic potential in patients with infertility and increased incidence of aneuploidy and to suppress impaired maturation of zygotes frequently observed in childbearing at an advanced age.