Gentisic acid attenuates 5-fluorouracil-induced ovotoxicity in rats via modulating Nrf2 signalling: An experimental approach.

5-Fluorouracil (5-FU) is the third most used chemotherapeutic in the world with its anticancer effect resulting from its potential to block DNA replication. Like other cytotoxic agents, 5-FU has side effects on healthy tissues, and the reproductive system is among the tissues most affected by these undesirable effects. Gentisic acid (GEA) is a secondary metabolite that is abundant in fruits, vegetables and spices and has antioxidant activity. This study was conducted to investigate the toxicity of 5-FU in rat ovarian tissue and to determine the therapeutic activity of GEA on ovotoxicity caused by 5-FU. The results showed that 5-FU caused histopathological findings by suppressing Nrf2 pathway and accordingly increasing oxidative stress, inflammation, endoplasmic reticulum stress and apoptosis. However, GEA treatments after 5-FU application ameliorated 5-FU-induced ovotoxicity dose-dependently through activation of Nrf2 pathway. All these findings provided strong evidence supporting the hypothesis that GEA treatment may have therapeutic effects against 5-FU-induced ovarian damage. However, the beneficial effect of GEA use in eliminating ovarian damage in women after 5-FU chemotherapy should continue to be investigated with more detailed molecular studies.

Gynotoxic Effects of Chemotherapy and Potential Protective Mechanisms.

Chemotherapy is one of the leading cancer treatments. Unfortunately, its use can contribute to several side effects, including gynotoxic effects in women. Ovarian reserve suppression and estrogen deficiency result in reduced quality of life for cancer patients and are frequently the cause of infertility and early menopause. Classic alkylating cytostatics are among the most toxic chemotherapeutics in this regard. They cause DNA damage in ovarian follicles and the cells they contain, and they can also induce oxidative stress or affect numerous signaling pathways. In vitro tests, animal models, and a few studies among women have investigated the effects of various agents on the protection of the ovarian reserve during classic chemotherapy. In this review article, we focused on the possible beneficial effects of selected hormones (anti-Müllerian hormone, ghrelin, luteinizing hormone, melatonin), agents affecting the activity of apoptotic pathways and modulating gene expression (C1P, S1P, microRNA), and several natural (quercetin, rapamycin, resveratrol) and synthetic compounds (bortezomib, dexrazoxane, goserelin, gonadoliberin analogs, imatinib, metformin, tamoxifen) in preventing gynotoxic effects induced by commonly used cytostatics. The presented line of research appears to provide a promising strategy for protecting and/or improving the ovarian reserve in the studied group of cancer patients. However, well-designed clinical trials are needed to unequivocally assess the effects of these agents on improving hormonal function and fertility in women treated with ovotoxic anticancer drugs.

The Protective Role of Magnoliae Flos in Preventing Ovotoxicity and Managing Ovarian Function: An In Vitro and In Vivo Study.

Magnoliae Flos (MF) is a medicinal herb widely employed in traditional medicine for relieving sinusitis, allergic rhinitis, headaches, and toothaches. Here, we investigated the potential preventive effects of MF extract (MFE) against 4-vinylcyclohexene diepoxide (VCD)-induced ovotoxicity in ovarian cells and a mouse model of premature ovarian insufficiency (POI). The cytoprotective effects of MFE were assessed using CHO-K1 or COV434 cells. In vivo, B6C3F1 female mice were intraperitoneally injected with VCD for two weeks to induce POI, while MFE was orally administered for four weeks, beginning one week before VCD administration. VCD led to a significant decline in the viabilities of CHO-K1 and COV434 cells and triggered excessive reactive oxygen species (ROS) production and apoptosis specifically in CHO-K1 cells. However, pretreatment with MFE effectively prevented VCD-induced cell death and ROS generation, while also activating the Akt signaling pathway. In vivo, MFE increased relative ovary weights, follicle numbers, and serum estradiol and anti-Müllerian hormone levels versus controls under conditions of ovary failure. Collectively, our results demonstrate that MFE has a preventive effect on VCD-induced ovotoxicity through Akt activation. These results suggest that MFE may have the potential to prevent and manage conditions such as POI and diminished ovarian reserve.

Syringic acid alleviates cisplatin-induced ovarian injury through modulating endoplasmic reticulum stress, inflammation and Nrf2 pathway.

BACKGROUND: Reproductive toxicity is one of the most important side effects of cisplatin (CIS) and leading to discontinuation of treatment. Syringic acid (SA) is a phenolic acid whose industrial use has increased in recent years due to its antioxidant properties. Recent reports highlight the importance of the supressed Nrf2 pathway in the molecular pathogenesis of CIS toxicity. Therefore, this study aimed to evaluate the therapeutic effect of SA on CIS-induced ovotoxicity through the Nrf2 pathway for the first time. MATERIAL AND METHODS: Thirty female rats were divided into 5 groups: control, CIS, CIS+SA (5 and 10 mg/kg) and only SA (per se, 10 mg/kg). CIS was administered intraperitoneally at a dose of 5 mg/kg on the 1st day, injections of SA followed by three consecutive days in the rats. Serum anti-mullerian hormone (AMH) levels and ovarian oxidative stress (OS), inflammation, endoplasmic reticulum stress (ERS), apoptosis and Nrf2 pathway markers were determined colorimetrically. Histopathological examinations of the ovaries with hematoxylin and eosin staining were also used to evaluate CIS-induced ovotoxicity. RESULTS: The CIS treatment depleted serum AMH levels, caused histopathological findings and increased OS, inflammation, ERS and apoptosis levels in ovarian tissue. However, treatments with SA significantly ameliorated CIS-induced biochemical and histopathological changes by activating Nrf2 pathway. CONCLUSION: The promising adjuvant potential of SA to alleviate CIS-related ovarian damage should be supported by more comprehensive studies.

Evaluation of the therapeutic effects of arbutin on cisplatin-induced ovarian toxicity in rats through endoplasmic reticulum stress and Nrf2 pathway.

Arbutin (ARB) is a glycosylated hydroquinone with potent antioxidant effects. Although cisplatin (CP) is widely used in chemotherapy, its toxicity in healthy tissues, including ovotoxicity, is an insurmountable problem. This study aimed to evaluate the therapeutic effect of ARB against CP-related ovototoxicity by including nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in rats for the first time. Rats treated one dose of CP (5 mg/kg) on the first day, followed by ARB (5 and 10 mg/kg) for three days. Serum reproductive hormone levels were determined using ELISA kits. Oxidative stress (OS), inflammation, endoplasmic reticulum stress (ERS) and apoptosis markers in ovarian tissue were also determined colorimetrically. In addition, how CP affects Nrf2 pathway and the effect of ARB on this situation were also addressed. ARB treatment reduced the levels of markers of OS, inflammation, ERS and apoptosis in ovarian tissue of CP-stimulated animals. ARB regenerated the depleted antioxidant system by triggering Nrf2 pathway in the ovarian tissues of animals stimulated by CP. Histological findings also supported the therapeutic efficacy of ARB. The results indicate that ARB may have therapeutic effects against CP-induced reproductive toxicity with its Nrf2 activator potential. ARB should be tested in more extensive studies as a new generation chemopreventive candidate molecule.

Ameliorative effect of gallic acid on cisplatin-induced ovarian toxicity in rats.

The aim of the present study was to evaluate the protective effect of gallic acid (GA) against cisplatin (CDDP)-induced ovarian toxicity, for the first time. The ovarian damage was generated with CDDP (5 mg/kg) intraperitoneally (i.p.) administration in rats. GA (2.5 and 5 mg/kg) were administered i.p. for 3 consecutive days. The study was carried out in 5 main groups containing 6 rats in each group: control, GA (5 mg/kg), CDDP, CDDP + GA (2.5 mg/kg) and CDDP + GA (5 mg/kg). The levels of ovarian malondialdehyde (MDA), total oxidant status (TOS), total antioxidant status (TAS), oxidative stress index (OSI), catalase (CAT), 8-hydroxy-2'-deoxyguanosine (8-OHdG), caspase-3 and tumor necrosis factor-alpha (TNF-α) were determined. Hematoxylin and eosin staining method was employed for the histopathological examination. In the CDDP group, it is determined that statistically significant decreasing in the levels of TAS and CAT, and increasing in the levels of MDA, TOS, OSI, 8-OHdG, caspase-3 and TNF-α (p < 0.05) compared with control group. GA administrations statistically significantly restored this damage (p < 0.05). Although vascular congestion, edema, hemorrhage, follicular degeneration and leukocyte infiltration were significantly higher in the CDDP group than in the control group, GA administrations statistically significantly restored these damages (p < 0.05). In conclusion, this study showed that GA prevented CDDP-induced ovarian damage with its antioxidant, anti-apoptotic and anti-inflammatory activities. More comprehensive studies are needed to see the underlying mechanisms.

Suppression of cisplatin-induced ovarian injury in rats by chrysin: an experimental study.

The aim of this study was to investigate the potential therapeutic efficacy of chrysin (CHS) against ovotoxicity caused by intraperitoneal administration of cisplatin (CDDP) in rats. In this experimental study, 24 female rats were randomly divided into four groups: control, CHS (2 mg/kg), CDDP (5 mg/kg) and CDDP (5 mg/kg) + CHS (2 mg/kg). The levels of malondialdehyde (MDA), total oxidant status (TOS), total antioxidant status (TAS), superoxide dismutase (SOD), interleukin-6 (IL-6) and myeloperoxidase (MPO) were determined in the ovarian tissues using spectrophotometric methods. In addition, the ovarian samples were evaluated histopathologically by hematoxylin&eosin staining. The results revealed that the levels of MDA, TOS, IL-6 and MPO significantly increased by CDDP administration compared with control group (p < 0.05). Also, it was found that CDDP significantly decreased TAS and SOD levels (p < 0.05). CHS ameliorated CDDP-induced the increased levels of MDA, TOS, IL-6, MPO and increased the levels of TAS and SOD significantly (p < 0.05). Histological findings also supported the therapeutic effect of CHS against CDDP-induced ovarian damage parameters. In conclusion, our results showed that CHS exhibits a therapeutic effect against CDDP-induced ovotoxicity and therefore the use of CHS after chemotherapy may improve the side effets of CDDP. IMPACT STATEMENTWhat is already known about this subject? Cisplatin (CDDP) is an effective and widely used chemotherapeutic agent to treat various malignancies, but its therapeutic use is limited due to dose-related tissue toxicity. Chrysin (CHS), a natural flavone, exhibits various beneficial activities, including antioxidant, anti-inflammatory and anticancer. There are increasing evidences in the literature that CHS reduces the toxicity of various chemotherapeutic agents, such as CDDP, doxorubicin and cyclophosphamide, in colon, kidney and liver tissues through its antioxidant and anti-inflammatory potential.What do the results of this study add? This study demonstrated that CHS can abolish CDDP-induced in vivo ovarian injury by decreasing MDA, TOS, IL-6 and MPO levels and increasing SOD and TAS levels through its antioxidant and anti-inflammatory potential.What are the implications of these findings for clinical practice and/or further research? This study revealed the therapeutic potential of CHS against CDDP-induced acute ovotoxicity, for the first time. Further pre-clinical studies are necessary to prove the beneficial effect of CHS on the prevention of CDDP-induced ovarian toxicity.

p-Coumaric acid alleviates cisplatin-induced ovarian toxicity in rats.

OBJECTIVE: The therapeutic value of cisplatin (CDDP) as an anticancer drug is limited by its ovo-otoxicity. The effect of natural phenolic acids in the prevention of many diseases related to oxidative stress has been reported. Here, the ability of p-coumaric (pCA) acid, a member of phenolic acids, to protect rat ovary tissue against CDDP-induced oxidative stress was investigated. METHODS: The study was carried out in five main groups containing six rats in each group: control, pCA (4 mg/kg), CDDP, CDDP plus pCA (2 mg/kg), and CDDP plus pCA (4 mg/kg). The levels of ovarian malondialdehyde (MDA), total oxidant status (TOS), total antioxidant status (TAS), oxidative stress index (OSI), catalase (CAT), 8-hydroxy-2'-deoxyguanosine (8-OHdG), caspase-3, and tumor necrosis factor-alpha (TNF-α) were determined. Hematoxylin and eosin staining method was employed for the histopathological examination. RESULTS: In the CDDP group, it is determined that statistically significant decreasing in the levels of TAS and CAT, and increasing in the levels of MDA, TOS, OSI, 8-OHdG, caspase-3, and TNF-α compared with control group (p < 0.05). pCA administration statistically significantly restored this damage in a dose-dependent manner (p < 0.05). Although vascular congestion, edema, hemorrhage, follicular degeneration, and leukocyte infiltration were significantly higher in the CDDP group than in the control group, pCA administrations statistically significantly restored these damages (p < 0.05). CONCLUSIONS: The data presented here indicate that pCA protects ovarian tissues of rats against CDDP-induced oxidative stress, inflammation, and apoptosis. It may be worthy to consider the usefulness of pCA as adjuvant therapy in cancer management.

Corrigendum: Hormone-Like Effects of 4-Vinylcyclohexene Diepoxide on Follicular Development.

[This corrects the article DOI: 10.3389/fcell.2020.00587.].

Hormone-Like Effects of 4-Vinylcyclohexene Diepoxide on Follicular Development.

BACKGROUND: 4-vinylcyclohexene diepoxide (VCD) has long been considered a hazardous occupational chemical that promotes ovarian failure. However, VCD is also used as a research compound to chemically induce animal models of premature ovarian insufficiency (POI), and in related work we unexpectedly found that VCD apparently exhibits both dose- and duration-dependent opposing, hormone-like effects on the maintenance of the primordial follicle pool, follicle development, and ovulation induction. RESULTS: We conducted experiments with cultured murine ovaries and performed transplantation experiments using postnatal day (PD) 2 and PD12 mice and found that low-dose, short-term exposure to VCD (VCDlow) actually protects the primordial/primary follicle pool and improves the functional ovarian reserve (FOR) by disrupting follicular atresia. VCDlow inhibits follicular apoptosis and regulates the Pten-PI3K-Foxo3a pathway. Short-term VCD exposure in vivo (80 mg/kg, 5 days) significantly increases the number of superovulated metaphase II oocytes, preovulatory follicles, and corpus luteum in middle-aged mice with diminished ovarian reserve (DOR). We demonstrate that low-dose but not high-dose VCD promotes aromatase levels in granulosa cells (GCs), thereby enhancing the levels of estradiol secretion. CONCLUSION: Our study illustrates a previously unappreciated, hormone-like action for the occupational "ovotoxin" molecule VCD and strongly suggests that VCDlow should be explored for its potential utility for treating human ovarian follicular development disorders, including subfertility in perimenopausal women.

A Tiered Female Ovarian Toxicity Screening Identifies Toxic Effects of Checkpoint Kinase 1 Inhibitors on Murine Growing Follicles.

Ovarian toxicity (ovotoxicity) is one of the major side effects of pharmaceutical compounds for women at or before reproductive age. The current gold standard for screening of compounds' ovotoxicity largely relies on preclinical investigations using whole animals. However, in vivo models are time-consuming, costly, and harmful to animals. Here, we developed a 3-tiered ovotoxicity screening approach starting from encapsulated in vitro follicle growth (eIVFG) and screened for the potential ovotoxicity of 8 preclinical compounds from AstraZeneca (AZ). Results from Tiers 1 to 2 screenings using eIVFG showed that the first 7 tested AZ compounds, AZ-A, -B, -C, -D, -E, -F, and -G, had no effect on examined mouse follicle and oocyte reproductive outcomes, including follicle survival and development, 17ß-estradiol secretion, ovulation, and oocyte meiotic maturation. However, AZ-H, a preclinical compound targeting the checkpoint kinase 1 inhibitor to potentiate the anticancer effects of DNA-damaging agents, significantly promoted granulosa cell apoptosis and the entire growing follicle atresia at clinically relevant concentrations of 1 and 10 µM. The more targeted explorations in Tier 2 revealed that the ovotoxic effect of AZ-H primarily resulted from checkpoint kinase 1 inhibition in granulosa cells. Using in vivo mouse model, the Tier 3 screening confirmed the in vitro ovotoxicities of AZ-H discovered in Tiers 1 and 2. Also, although AZ-H at 0.1 µM alone was not ovotoxic, it significantly exacerbated gemcitabine-induced ovotoxicities on growing follicles. Taken together, our study demonstrates that the tiered ovotoxicity screening approach starting from eIVFG identifies and prioritizes pharmaceutical compounds of high ovotoxicity concern.

A closed vitrification system enables a murine ovarian follicle bank for high-throughput ovotoxicity screening, which identifies endocrine disrupting activity of microcystins.

Increasing evidence reveals that a broad spectrum of environmental chemicals and pharmaceutical compounds cause female ovarian toxicity (ovotoxicity). The current gold standard of ovotoxicity testing largely relies on whole laboratory animals, but in vivo models are time consuming, costly, and present animal welfare concerns. We previously demonstrated that the 3D encapsulated in vitro follicle growth (eIVFG) is a robust in vitro model for ovotoxicity testing. However, the follicle preparation process is complex and highly dependent on technical skills. Here, we aimed to use vitrification method to cryopreserve murine immature follicles for a high-content eIVFG, chemical exposure, and ovotoxicity screening. Results indicated that a closed vitrification system combined with optimized vitrification protocols preserved mouse follicle viability and functionality and vitrified follicles exhibited comparable follicle and oocyte reproductive outcomes to freshly harvested follicles during eIVFG, including follicle survival and development, ovarian steroidogenesis, and oocyte maturation and ovulation. Moreover, vitrified follicles consistently responded to ovotoxic chemical, doxorubicin (DOX). We further used vitrified follicles to test the response of microcystins (MCs), an emerging category of environmental contaminants produced by cyanobacteria associated with harmful algal blooms (HABs), and found that different congeners of MCs exhibited differential ovotoxicities. In summary, our study demonstrates that vitrification enables a long-term-storage and ready-to-use ovarian follicle bank for high-throughput ovotoxicity screening, which identifies endocrine disrupting effects of MCs.

Ataxia-telangiectasia mutated coordinates the ovarian DNA repair and atresia-initiating response to phosphoramide mustard.

Ataxia-telangiectasia-mutated (ATM) protein recognizes and repairs DNA double strand breaks through activation of cell cycle checkpoints and DNA repair proteins. Atm gene mutations increase female reproductive cancer risk. Phosphoramide mustard (PM) induces ovarian DNA damage and destroys primordial follicles, and pharmacological ATM inhibition prevents PM-induced follicular depletion. Wild-type (WT) C57BL/6 or Atm+/- mice were dosed once intraperitoneally with sesame oil (95%) or PM (25 mg/kg) in the proestrus phase of the estrous cycle and ovaries harvested 3 days thereafter. Atm+/- mice spent ~25% more time in diestrus phase than WT. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) on ovarian protein was performed and bioinformatically analyzed. Relative to WT, Atm+/- mice had 64 and 243 proteins increased or decreased in abundance, respectively. In WT mice, PM increased 162 and decreased 20 proteins. In Atm+/- mice, 173 and 37 proteins were increased and decreased, respectively, by PM. Exportin-2 (XPO2) was localized to granulosa cells of all follicle stages and was 7.2-fold greater in Atm+/- than WT mice. Cytoplasmic FMR1-interacting protein 1 was 6.8-fold lower in Atm+/- mice and was located in the surface epithelium with apparent translocation to the ovarian medulla post-PM exposure. PM induced γH2AX, but fewer γH2AX-positive foci were identified in Atm+/- ovaries. Similarly, cleaved caspase-3 was lower in the Atm+/- PM-treated, relative to WT mice. These findings support ATM involvement in ovarian DNA repair and suggest that ATM functions to regulate ovarian atresia.

Cypermethrin exposure reduces the ovarian reserve by causing mitochondrial dysfunction in granulosa cells.

Cypermethrin (CYP) is one of the most highly effective synthetic pyrethroid insecticides and is recommended for insect control because it is considered to be relatively non-toxic to humans in all stages of life. However, recent data have shown that CYP has adverse effects on fertility, immune system, cardiovascular, hepatic metabolism and enzyme activity in vertebrates. Our objective was to investigate the toxicity of CYP to the ovary and to elucidate the underlying molecular mechanisms. Twenty 8-week-old CD-1 female mice were randomly assigned to four groups, that were separately exposed to CYP at doses of 12.5, 25 and 50 mg/kg/day or to corn oil (vehicle) for 28 days by intragastric administration. Moreover, human granulosa KGN cells were treated with CYP for 24 h at concentrations of 100 µM and 200 µM or to anhydrous ethanol (vehicle). This study clearly demonstrated that, compared to vehicle exposure, CYP exposure caused abnormal estrous cyclicity and decreased follicle numbers in all stages of mice with a dose-dependent manner. Interestingly, the apoptosis signals were mainly located in granulosa cells, and the expression levels of Caspase 3, Bax and Bcl-2 were increased in the 25 and 50 mg/kg/d groups. In KGN cells, CYP (100 and 200 µM) clearly induced apoptosis together with mitochondrial membrane potential depolarization and abnormal ROS generation compared to the control group. Altogether, these results suggest that CYP exposure reduced the ovarian reserve in mice by inducing apoptosis in granulosa cells via mitochondrial-related pathways.

Vincristine Chemotherapy Induces Atresia of Growing Ovarian Follicles in Mice.

With great advances in cancer detection and treatment, patient survival rates have improved substantially. Subsequently, significant efforts are now focused on improving the long-term sequelae of anticancer therapies in survivors, which includes fertility. Vincristine is a microtubule destabilizing antimitotic chemotherapeutic agent commonly administered for the treatment of cancers or autoimmune disorders prevalent in girls and women of reproductive age. The potential off-target effects of vincristine on the ovary have not been directly examined. Eight-week and 6-month-old C57BL/6J mice were administered with vincristine (1 mg/kg/bw/day) or saline on day (d)1, d4, and d8, then sacrificed after 24 hours (h), or 14 days (n = 4-6/group). We assessed the impact of vincristine on the ovarian reserve of quiescent primordial follicles, as well as growing follicles, which produce mature ovulatory oocytes. This study clearly demonstrated that multidose vincristine administration caused acute atresia and loss of growing follicles and reduced corpora luteua counts 24 h following final treatment. Treatment also disrupted estrous cycling and reduced serum anti-Müllerian hormone levels. However, primordial follicle numbers were unaffected, and growing follicle populations were restored to control levels 14 days after final treatment. Vincristine exerted similar effects on ovarian follicle populations in both 8-week-old reproductively young mice and reproductively older 6-month-old mice. This study suggests that vincristine, administrated at the current dose, is toxic to growing follicles but does not deplete primordial follicles in mice. Further studies should be performed before extrapolating these data to infer the consequences of vincristine on the ovary in humans.

Obesity alters phosphoramide mustard-induced ovarian DNA repair in mice.

Phosphoramide mustard (PM) destroys rapidly dividing cells and activates the DNA double strand break marker, γH2AX, and DNA repair in rat granulosa cells and neonatal ovaries. The effects of PM exposure on DNA damage and activation of DNA damage repair in lean and obese female mice were investigated. Wild type (lean) non agouti (a/a) and KK.Cg-Ay/J heterozygote (obese) mice received sesame oil or PM (95%; 25 mg/kg; intraperitoneal injection). Obesity increased (P < 0.05) hepatic and spleen but decreased (P < 0.05) uterine weight. PM exposure reduced (P < 0.05) spleen weight regardless of body composition, however, decreased (P < 0.05) ovarian and hepatic weight were observed in the obese PM-exposed females. PM decreased (P < 0.05) primordial and primary follicle number in lean females. Obesity and PM increased (P < 0.05) γH2AX protein. DNA damage repair genes Prkdc, Parp1, and Rad51 mRNA were unaltered by obesity, however, Atm and Xrcc6 mRNA were increased (P < 0.05) while Brca1 was reduced (P < 0.05). Obesity reduced (P < 0.05) PRKDC, XRCC6 and but increased (P < 0.05) ATM protein. ATM, BRCA1 and RAD51 protein levels were increased (P < 0.05) by PM exposure in both lean and obese mice, while PM-induced increased (P < 0.05) XRCC6 and PARP1 were observed only in lean mice. Thus, PM induces ovarian DNA damage in vivo; obesity alters DNA repair response gene mRNA and protein level; the ovary activates DNA repair proteins in response to PM; but obesity compromises the ovarian PM response.

Phosphoramide mustard induces autophagy markers and mTOR inhibition prevents follicle loss due to phosphoramide mustard exposure.

Phosphoramide mustard (PM) is an ovotoxic metabolite of cyclophosphamide. Postnatal day 4 Fisher 344 rat ovaries were exposed to vehicle control (1% DMSO) or PM (60µM)±LY294002 or rapamycin for 2 or 4 d. Transmission election microscopy revealed abnormally large golgi apparatus and electron dense mitochondria in PM-exposed ovaries prior to and at the time of follicle depletion. PM exposure increased (P<0.05) mRNA abundance of Bbc3, Cdkn1a, Ctfr, Edn1, Gstp1, Nqo1, Tlr4, Tnfrsfla, Txnrd1 and decreased (P<0.05) Casp1 and Il1b after 4d. PM exposure increased (P<0.1) BECN1 and LAMP, decreased (P<0.1) ABCB1 and did not alter ABCC1 protein. LY294002 did not impact PM-induced ovotoxicity, but decreased ABCC1 and ABCB1 protein. Rapamycin prevented PM-induced follicle loss. These data suggest that the mammalian target of rapamycin, mTOR, may be a gatekeeper of PM-induced follicle loss.

Akt activation by Evodiae Fructus extract protects ovary against 4-vinylcyclohexene diepoxide-induced ovotoxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: Evodiae Fructus (EF) is the dried, unripe fruit of Evodia rutaecarpa Benth., and one of the main components of traditional herbal prescriptions issued for the treatment of sterility caused by irregular menstruation in Korea. However, scientific evidence regarding the efficacy and action mechanism of EF is lacking. AIM OF THE STUDY: In this study, the authors established an in vitro screening tool to identify promising new drug candidates in herbal medicines for the prevention and treatment of premature ovarian failure. The protective effects of EF extracts against 4-vinylcyclohexene diepoxide (VCD)-induced ovotoxicity were investigated and the molecular mechanism responsible was sought. MATERIAL AND METHODS: EF extract was prepared by boiling EF in water and its quality was confirmed by high performance liquid chromatography. CHO-K1 (Chinese hamster ovary cells) and COV434 (human ovarian granulosa cells) cells were plated, pretreated with EF extract for 2h and then treated with 1.5mM or 0.5mM VCD for 24h, respectively. Cell viabilities were measured using an MTT assay, and protein levels were determined by western blotting. RESULTS: VCD significantly suppressed the viability of both CHO-K1 and COV434 cells in a dose-dependent manner and induced the apoptosis of CHO-K1 cells at 1.5mM. EF extract dose-dependently blocked the ovotoxicity induced by treatment with VCD. Furthermore, EF extract significantly activated Akt and downstream effectors such as mTOR and GSK-3ß in CHO-K1 cells. The ability of EF extract to prevent cytotoxicity by VCD was antagonized by pretreatment of LY294002, a PI3K/Akt inhibitor. CONCLUSION: EF has the ability to protect ovary cells against VCD-induced ovotoxicity, probably via Akt activation. These results suggest that the beneficial effects of EF might be useful for preventing premature ovarian failure or unexplained infertility caused by environmental factors.

Enhanced susceptibility of ovaries from obese mice to 7,12-dimethylbenz[a]anthracene-induced DNA damage.

7,12-Dimethylbenz[a]anthracene (DMBA) depletes ovarian follicles and induces DNA damage in extra-ovarian tissues, thus, we investigated ovarian DMBA-induced DNA damage. Additionally, since obesity is associated with increased offspring birth defect incidence, we hypothesized that a DMBA-induced DNA damage response (DDR) is compromised in ovaries from obese females. Wild type (lean) non agouti (a/a) and KK.Cg-Ay/J heterozygote (obese) mice were dosed with sesame oil or DMBA (1mg/kg; intraperitoneal injection) at 18weeks of age, for 14days. Total ovarian RNA and protein were isolated and abundance of Ataxia telangiectasia mutated (Atm), X-ray repair complementing defective repair in Chinese hamster cells 6 (Xrcc6), breast cancer type 1 (Brca1), Rad 51 homolog (Rad51), poly [ADP-ribose] polymerase 1 (Parp1) and protein kinase, DNA-activated, catalytic polypeptide (Prkdc) were quantified by RT-PCR or Western blot. Phosphorylated histone H2AX (γH2AX) level was determined by Western blotting. Obesity decreased (P<0.05) basal protein abundance of PRKDC and BRCA1 proteins but increased (P<0.05) γH2AX and PARP1 proteins. Ovarian ATM, XRCC6, PRKDC, RAD51 and PARP1 proteins were increased (P<0.05) by DMBA exposure in lean mice. A blunted DMBA-induced increase (P<0.05) in XRCC6, PRKDC, RAD51 and BRCA1 was observed in ovaries from obese mice, relative to lean counterparts. Taken together, DMBA exposure induced γH2AX as well as the ovarian DDR, supporting that DMBA causes ovarian DNA damage. Additionally, ovarian DDR was partially attenuated in obese females raising concern that obesity may be an additive factor during chemical-induced ovotoxicity.

Raloxifene protects against seizures and neurodegeneration in a mouse model mimicking epilepsy in postmenopausal woman.

Epilepsy in menopausal women presents several challenges in the treatment including an increased risk of seizures due to hormone replacement therapy. We investigated the hypothesis if raloxifene, a selective oestrogen receptor modulator, could be employed to prevent behavioural seizures and morphological alterations in a mouse model mimicking epilepsy in postmenopausal women. Female mice were made ovotoxic by treatment with 4-vinylcyclohexene diepoxide (VCD) to mimic a postmenopausal state. They were then subjected to kainic acid (KA)-induced seizures and neurotoxicity, as assessed by microscopic examination of hippocampus, relevant to human temporal lobe epilepsy. VCD administration (for 15days followed by a drug-free period of 30days) induced ovotoxicity in mice as evidenced by reduced number of primary ovarian follicles. This was accompanied by a 62.4% reduction in serum oestradiol levels. The bone mineral density of ovotoxic mice, however, remained unaffected. Raloxifene (8mg/kg) reduced the seizure severity score in both normal and ovotoxic mice and protected against degeneration induced by KA in the CA3, CA1 sub-fields and hilus of the DG. Hippocampal TGF-ß3 levels were not affected by any of the treatments. We show the potential protective role of raloxifene in preventing seizures and neuronal damage in a mouse model mimicking epilepsy in postmenopausal women which was found unrelated to hippocampal TGF-ß3. Raloxifene might represent a novel therapeutic option for postmenopausal temporal lobe epileptic woman.