Persistent follicular granulosa cell senescence and apoptosis induced by methotrexate leading to oocyte dysfunction and aberrant embryo development.

Evidence from clinical cases indicates an association between the low success rate of in vitro fertilization (IVF) and ovarian injury due to previous methotrexate (MTX) administration. Therefore, it is necessary to develop and propose reasonable clinical drug guidelines to improve the quality of oocytes and the development of embryos before pregnancy. In this study, we established a mouse model with previous MTX exposure to validate the effects of MTX on reproductive function in female mice. We observed that MTX administration could result in a decrease in the success rate of fertilization and an aberrant embryonic development in both natural fertilization and IVF, even after completion of five to six ovulation cycles after MTX withdrawal. Further research revealed senescence and apoptosis of follicular granulosa cells (GCs), accompanied by arrested follicle development and aberrant estradiol and anti-Mullerian hormone levels. Supportive evidence indicated that MTX administration induced senescence and apoptosis of human GCs in vitro, and the effects were consistent with the high levels of p21, p53, and oxidative stress. We further demonstrated that folic acid (FA) could improve oocyte function and embryonic development in vivo and in vitro by protecting GCs against apoptosis and senescence. Based on these findings, we propose the implementation of extended intervals between MTX exposure and conception or IVF and recommend FA as a special dietary supplement during this interval period; however, prospective inquiry in humans is necessary to further understand the relationship between MTX and FA recovery.

Induction of Oxidative Stress and Mitochondrial Dysfunction by Juglone Affects the Development of Bovine Oocytes.

Juglone, a major naphthalenedione component of walnut trees, has long been used in traditional medicine as an antimicrobial and antitumor agent. Nonetheless, its impact on oocyte and preimplantation embryo development has not been entirely clarified. Using the bovine model, we sought to elucidate the impact of juglone treatment during the in vitro maturation (IVM) of oocytes on their maturation and development of embryos. Results showed a severe reduction in oocyte nuclear maturation and cumulus expansion and a significant increase in mitochondrial dysfunction and reactive oxygen species (ROS) levels in cumulus-oocyte complexes (COCs) treated with juglone (12.5, 25.0, and 50.0 µM). In addition, RT-qPCR showed downregulation of the expansion-related (HAS2, TNFAIP6, PTX3, and PTGS2) and mitochondrial (ATPase6 and ATP5F1E) genes in juglone-treated COCs. Moreover, the development rates of day 4 total cleavage and 8-16 cell stage embryos, as well as day 8 blastocysts, were significantly reduced following exposure to juglone. Using immunofluorescence, the apoptotic marker caspase-9 was overexpressed in oocytes exposed to juglone (25.0 µM) compared to the untreated control. In conclusion, our study reports that exposing bovine oocytes to 12.5-50.0 µM of juglone can reduce their development through the direct induction of ROS accumulation, apoptosis, and mitochondrial dysfunction.

Samul-tang ameliorates oocyte damage due to cyclophosphamide-induced chronic ovarian dysfunction in mice.

Samul-tang (SM), a traditional herbal medicine, has been used to treat menstrual irregularities and infertility in women. However, the cellular and molecular mechanisms underlying the effects of SM remain elusive. We investigated the potential protective effect of SM against chronic ovarian dysfunction and used bioinformatics analysis to identify its underlying mechanism in a mouse model of cyclophosphamide (CP)-induced diminished ovarian reserve. Female C57BL/6 mice were intraperitoneally injected with CP three times a week, followed by oral administration of distilled water (CP group) or SM (CP + SM group) for 4 weeks. Four weeks later, the effect of SM was assessed by ovarian tissue histological analysis, steroid hormone measurement, oocyte quality, and mRNA and microRNA microarray analysis in the ovaries. Although SM administration did not prevent CP-induced follicle loss in mice, the quality of oocytes was better in CP + SM mice than in CP mice. Gene expression analysis revealed that the expression of fertilisation- and ovarian follicle development-related genes was altered by CP treatment but normalized after SM administration. Further bioinformatics analysis showed possible interactions between differentially expressed mRNAs and microRNAs. Therefore, we demonstrated the protective effects of SM on ovarian function and oocyte maturation against CP-induced damage via multiple epigenetic mechanisms.

Coenzyme Q10 supplementation of human oocyte in vitro maturation reduces postmeiotic aneuploidies.

OBJECTIVE: To evaluate the effect of coenzyme Q10 (CoQ10) supplementation on oocyte maturation rates and postmeiotic aneuploidy rates during in vitro maturation (IVM) of human oocytes. DESIGN: Clinical laboratory observation. SETTING: Hospital and university laboratories. PATIENT(S): Forty-five patients aged ≥38 years and 18 patients aged ≤30 years undergoing in vitro fertilization. INTERVENTION(S): The germinal vesicle-stage oocytes and associated cumulus cells were cultured in IVM media for 24-48 hours with or without 50 µmol/L CoQ10. Oocyte maturation rates were determined based on the presence or absence of the first polar body. Postmeiotic aneuploidies were determined using next-generation sequencing analyses of biopsied polar bodies. MAIN OUTCOME MEASURE(S): Oocyte maturation rates, postmeiotic oocyte aneuploidy rates, and chromosome aneuploidy frequencies. RESULT(S): In women aged 38-46 years, 50 µmol/L CoQ10 significantly increased oocyte maturation rates (82.6% vs. 63.0%; P=.035), reduced oocyte aneuploidy rates (36.8% vs. 65.5%; P=.020), and reduced chromosome aneuploidy frequencies (4.1% vs. 7.0%; P=.012. In women aged ≤30 years, we failed to demonstrate an effect of CoQ10 on oocyte maturation rates or postmeiotic aneuploidies. CONCLUSION(S): CoQ10 supplementation during IVM increased oocyte maturation rates and reduced postmeiotic aneuploidies for older women.

Shoutai pills improve the quality of oocytes exposed to the chemotherapeutic drug Hydroxyurea.

Hydroxyurea (HU), a DNA synthesis inhibitor, is one of the most common chemotherapeutic drugs that have been widely applied to treat a variety of cancers. HU treatment exhibits severe side effects including renal toxicity, skin toxicity and embryo-toxicity. However, the influence of HU on the female gamete development has not yet fully clarified. Here, we found that HU exposure induced the degeneration of activated follicles after primordial follicle stage, resulting in the depletion of the ovarian reserve. HU exposure also led to the oocyte meiotic maturation arrest via disrupting normal spindle assembly, chromosome alignment and kinetochore-microtubule attachment. Furthermore, exposure to HU impaired the dynamics of ovastacin and Juno, two critical fertilization regulators. Notably, we illustrated that Shoutai pills (STP), a traditional Chinese medicine drug that has been commonly used for the treatment of miscarriage in China, partially restored all of the defects of oocyte development resulting from HU exposure through inhibiting the occurrence of oxidative stress-induced apoptosis. Taken together, our data not only reveal the adverse impact of HU exposure on the female gamete development, but also provide an effective strategy to prevent it, potentially contributing to the improvement of the quality of oocytes from patients treated with HU.

In vitro supplementation of trans-10, cis-12 conjugated linoleic acid ameliorated deleterious effect of heat stress on bovine oocyte developmental competence.

Environmental stresses, such as heat stress (HS), have been shown to have diverse effects on the developmental competence of oocytes. The aim of this study was to determine the effect of exogenous conjugated linoleic acid (CLA) supplementation in maturation medium on bovine oocyte maturation and developmental competence under HS condition. Accordingly, cumulus-oocyte complexes (COCs) were cultured at 41 °C and 38.5 °C for the first and second 12 h of maturation in the presence of 0 (PC), 50 (CLA50-HS) and 100 (CLA100-HS) µM CLA. Also, a group of COCs were cultured at 38.5 °C for 24 h of maturation without CLA supplementation as negative control (NC). Nuclear maturation, level of intracellular glutathione (GSH), reactive oxygen species (ROS) content, cleavage and blastocyst rates as well as relative expression of BAX, and BCL2 genes in blastocysts were investigated. Our finding for the PC and NC groups revealed that HS decreased the percentage of MII oocytes, cleavage and blastocyst rates (P < 0.05). Moreover, HS lead to an increase in ROS levels and relative expression of BAX gene, decreased the intracellular content of GSH and relative expression of BCL2 gene (P < 0.05). However, the cleavage and blastocyst rates tended to increase in the CLA-supplemented groups compared to PC group (p < 0.10). Also, ROS and GSH levels in the matured oocytes decreased and increased in the CLA50-HS group compared to the PC group (P < 0.05), respectively. The ratio of expression levels of BAX to BCL2 genes was not different between the PC and CLA50-HS groups (P > 0.05). These findings suggest that HS has undesirable effects on the maturation competence of bovine oocyte and subsequent embryo development while administration of CLA can ameliorate some of adverse effects of HS.

N-acetyl-L-cysteine (NAC) delays post-ovulatory oocyte aging in mouse.

The quality of post-ovulatory oocytes decreases with aging. In this study, we aimed to investigate the effects of N-acetyl-L-cysteine (NAC), a broadly used antioxidant, on oocyte quality in mouse post-ovulatory oocyte aging in vitro. NAC at 0.6mM concentration was added to culture medium (M2), and the quality of oocytes was analyzed at 6h, 12h, 18h and 24h of culture. We found that the frequency of spindle defects decreased in NAC-treated oocytes compared to those without NAC treatment. NAC treatment significantly decreased abnormal distribution of cortical granules (CGs) in oocytes during aging for 18h and 24h. Decreased intracellular reactive oxygen species (ROS) was also observed. Increased intracellular ATP levels and decreased abnormal distribution of mitochondria could be observed with NAC supplementation during post-ovulatory oocyte aging in vitro. These results indicate that NAC will maintain the quality of oocytes, and delay post-ovulatory oocyte aging as studied in the mouse.

Vitamin C protects against defects induced by juglone during porcine oocyte maturation.

Juglone, a naphthoquinone isolated from many species of the Juglandaceae family, has been used in traditional Chinese medicine for centuries because of its antiviral, antibacterial, and antitumor activities. However, the toxicity of juglone has also been demonstrated. Here, we used porcine oocytes as a model to explore the effects of juglone on oocyte maturation and studied the impact of vitamin C (VC) administration on juglone exposure-induced meiosis defects. Exposure to juglone significantly restricted cumulus cell expansion and decreased the first polar body extrusion. In addition, juglone exposure disturbed spindle organization, actin assembly, and the distribution of mitochondria during oocyte meiosis, while the acetylation level of α-tubulin was also reduced. These defects were all ameliorated by VC administration. Our findings indicate that juglone exposure induced meiotic failure in porcine oocytes, while VC protected against these defects during porcine oocyte maturation by ameliorating the organization of the cytoskeleton and mitochondrial distribution.

Melatonin alleviates the deterioration of oocytes from mice subjected to repeated superovulation.

Induction of repeated superovulation with exogenous hormones is widely used in assisted reproductive technology (ART). Though it is generally safe, emerging evidence has indicated that repeated superovulation may compromise oocyte quality. However, few studies have explored how to ameliorate such impairment. Because melatonin has beneficial influences on oocytes in various detrimental environments, we aimed to explore whether melatonin could protect mouse oocytes after repeated superovulation. We found that repeated superovulation markedly reduced meiotic maturation and disrupted spindle organization and chromosome alignment. Furthermore, we observed reduced mitochondrial content and enhanced early apoptosis in oocytes from mice subjected to repeated superovulation. In addition, 5-methylcytosine (5mc) fluorescence intensity was lower in oocytes from experimental mice than in those from control mice, indicating that repeated superovulation disrupts genomic DNA methylation, and elevations in reactive oxygen species levels indicated that repeated superovulation also induces oxidative stress. Conversely, melatonin administration improved oocyte maturation and attenuated the observed defects. Interestingly, supplementation with melatonin during in vitro maturation had the same protective effects on oocytes as in vivo melatonin administration. In summary, our results show that melatonin can improve oocyte quality after repeated superovulation and thus provide a potential strategy to improve ART efficiency.

Incidence of apoptosis after retinoids and insulin-like growth factor-I (IGF-I) supplementation during goat in vitro embryo production.

The addition of growth factors and vitamins enhances goat embryonic development in vitro. However, few attempts have been reported trying to identify supplementation regimens for oocyte maturation or embryo culture with additive properties. The present report was aimed to evaluate if retinoids [0.3 µM retinyl acetate (RAc) and 0.5 µM 9-cis-retinoic acid (RA)] supplementation during goat oocyte maturation and retinoids and/or 50 ng mL-1 IGF-I during embryo culture synergically enhanced embryonic development while diminishing the incidence of apoptosis. All combinations of RAc and RA treatment produced blastocysts with similar efficiencies, while IGF-I enhanced embryos yields irrespectively of retinoid addition. Moreover, retinoids and IGF-I supplementation showed similar caspase activity or DNA fragmentation indexes in blastocysts. In conclusion, supplementation with retinoids and IGF-I during goat embryo culture enhances blastocysts development without synergic reduction of apoptosis.

The effect of FT500 Plus(®) on ovarian stimulation in PCOS women.

Both oxidative stress and polycystic ovary syndrome have been involved in several aspects of female reproduction. In this retrospective observational study, the outcome of controlled ovarian stimulation and follicular microenvironment of twenty-five women affected by PCOS (Group A) have been explored, evaluating the effects of myo-inositol in association with antioxidant activities (FT500 Plus(®)). Twenty-five untreated-PCOS women (Group B) with similar characteristics served as control group. Although there was no difference in ovarian volume at time zero, this parameter was significantly smaller at the 5-month follow-up in the Group A (11.1±0.9 versus 13.5±1; P=0.0001). Group A showed a significant increase in the number of MII oocytes (6.3±2.5 versus 4.5±2; P=0.03) and glutathione peroxidase activity in follicular fluid (15.4±6.2 versus 11±2.2; P=0.04). FT500 Plus(®) may be considered in PCOS patient for improving oocyte quality.

A Systematic Review of the Molecular Mechanisms of Uranium -Induced Reproductive Toxicity.

Uranium is the heaviest metal known as nuclear fuel, and employed in the production of glass tinting compounds, ceramic glazes, gyroscope wheels, chemical catalysts and X-ray tube targets. Inhalation and ingestion are two of the most usual ways of exposure. Uranium may be released into drinking water through the mining leading to contamination. Uranium is able to damage the DNA by generation of free radicals and acting as a catalyst in the Fenton reactions causing oxidative stress. In fact, reproductive system contains high amount of polyunsaturated fatty acids, and therefore it is highly vulnerable to reactive oxygen species (ROS) and sensitive to uranium toxicity. Toxic effects of uranium are generally reported through different mechanisms of action including inflammation, degeneration of testis, vacuolization of Leydig cells, spermatocytes necrosis, and oocyte dysmorphism. The present article provides a comprehensive review of the recent findings mostly about the molecular and biochemical toxicity of uranium on the reproductive system.

Prooxidant effects of verbascoside, a bioactive compound from olive oil mill wastewater, on in vitro developmental potential of ovine prepubertal oocytes and bioenergetic/oxidative stress parameters of fresh and vitrified oocytes.

Verbascoside (VB) is a bioactive polyphenol from olive oil mill wastewater with known antioxidant activity. Oxidative stress is an emerging problem in assisted reproductive technology (ART). Juvenile ART is a promising topic because, in farm animals, it reduces the generation gap and, in human reproductive medicine, it helps to overcome premature ovarian failure. The aim of this study was to test the effects of VB on the developmental competence of ovine prepubertal oocytes and the bioenergetic/oxidative stress status of fresh and vitrified oocytes. In fresh oocytes, VB exerted prooxidant short-term effects, that is, catalase activity increase and uncoupled increases of mitochondria and reactive oxygen species (ROS) fluorescence signals, and long-term effects, that is, reduced blastocyst formation rate. In vitrified oocytes, VB increased ROS levels. Prooxidant VB effects in ovine prepubertal oocytes could be related to higher VB accumulation, which was found as almost one thousand times higher than that reported in other cell systems in previous studies. Also, long exposure times of oocytes to VB, throughout the duration of in vitro maturation culture, may have contributed to significant increase of oocyte oxidation. Further studies are needed to identify lower concentrations and/or shorter exposure times to figure out VB antioxidant effects in juvenile ARTs.

Effect of antifreeze protein supplementation in vitrification medium on mouse oocyte developmental competence.

OBJECTIVE: To investigate the effect of antifreeze protein (AFP) supplementation during mouse oocyte vitrification on the survival, fertilization and embryonic development. DESIGN: Animal study. SETTING: University laboratory. ANIMAL(S): BDF-1 mice. INTERVENTION(S): In vivo-matured metaphase II oocytes were vitrified with the use of CryoTop by two-step exposure to equilibrium and vitrification solution supplemented or not with 500 ng/mL AFP III. MAIN OUTCOME MEASURE(S): Postwarming survival, fertilization, embryonic development up to blastocyst in vitro, morphology of spindle and chromosome, membrane integrity, adenosine triphosphate (ATP) contents, and several gene expressions. RESULT(S): In the AFP-treated group, blastocyst formation rate was significantly higher and blastomere count with positive caspase was significantly lower compared with the nontreated group. Rate of intact spindle/chromosome, stable membrane, and ATP contents were significantly higher in AFP group. AFP group showed higher Mad2 and lower Eg5 gene expression. Both vitrification groups showed increased Hsf1, Zar1, and Zp1/Zp2 expression and decreased Hook1 and Zp3 expression compared with fresh control samples. CONCLUSION(S): Supplementation of AFP in vitrification medium has a protective effect on mouse oocytes for chilling injury; it can preserve spindle/membrane integrity and intracellular ATP contents. More stable spindle integrity in the AFP group may be associated with higher Mad2 and lower Eg5 gene expression.

Cadmium-induced ovarian pathophysiology is mediated by change in gene expression pattern of zinc transporters in zebrafish (Danio rerio).

This study explored the potential for expression pattern of genes encoding zinc (Zn) transporters to be involved in the cadmium (Cd)-induced reproductive toxicity in female of zebrafish. For this purpose, oocytes maturity and ovarian histology as well as Cd, Zn and metallothioneins (MTs) accumulation and expression of genes encoding Zrt-,Irt-related protein 10 (ZIP10), Zn transporter 1 (ZnT1) and zebrafish metallothionein (zMT) were examined in ovaries of adult zebrafish exposed to 0.4 mg/L Cd in water and supplemented with Zn (5 mgkg(-1)) in their diet for 21 days. Cd-exposure decreased the expression of ZnT1 and caused up-regulation of ZIP10 and zMT gene expression. These changes were accompanied by increased Cd and MTs accumulation, decreased Zn contents as well as by histopathological damages in ovarian tissues. The co-exposure of fish to Cd and Zn abolished ZnT1 down-regulation and rendered a persistently increased ZIP10 mRNA level. This treatment also decreased Cd and MTs accumulation, reversed Cd-induced Zn depletion and partially restored Cd-induced histological changes in ovarian tissues. These results imply that the downregulation of ZnT1 as well as the overexpression of ZIP10, in responses to the ovarian Zn depletion induced by Cd, play a major role in Cd accumulation and consequently in its toxicity. The protective effect of dietary Zn supplementation against Cd-induced toxicity is mediated, at least in part, by the increase of Zn availability and subsequently the induction of ZnT1 gene expression.

Miscarriage rates after dehydroepiandrosterone (DHEA) supplementation in women with diminished ovarian reserve: a case control study.

BACKGROUND: Dehydroepinadrosterone (DHEA) supplementation improves pregnancy chances in women with diminished ovarian reserve (DOR), by possibly reducing aneuploidy. Since a large majority of spontaneous miscarriages are associated with aneuploidy, one can speculate that DHEA supplementation may also reduce miscarriage rates. METHODS: We retroactively compared, utilizing two independent statistical models, miscarriage rates in 73 DHEA supplemented pregnancies at two independent North American infertility centers, age-stratified, to miscarriages reported in a national U.S. in vitro fertilization (IVF) data base. RESULTS: After DHEA supplementation the miscarriage rate at both centers was 15.1% (15.0% and 15.2%, respectively). For DHEA supplementation Mantel-Hänszel common odds ratio (and 95% confidence interval), stratified by age, was significantly lower, relative to odds of miscarriage in the general IVF control population [0.49 (0.25-0.94; p = 0.04)]. Miscarriage rates after DHEA were significantly lower at all ages but most pronounced above age 35 years. DISCUSSION: Since DOR patients in the literature are reported to experience significantly higher miscarriage rates than average IVF patients, the here observed reduction in miscarriages after DHEA supplementation exceeds, however, all expectations. Miscarriage rates after DHEA not only were lower than in an average national IVF population but were comparable to rates reported in normally fertile populations. Low miscarriage rates, comparable to those of normal fertile women, are statistically impossible to achieve in DOR patients without assumption of a DHEA effect on embryo ploidy. Beyond further investigations in infertile populations, these data, therefore, also suggest the investigations of pre-conception DHEA supplementation in normal fertile populations above age 35 years.

Injury effects of ginkgolide B on maturation of mouse oocytes, fertilization, and fetal development in vitro and in vivo.

Ginkgolide B (GKB), the major active component of Ginkgo biloba extracts, exerts both stimulatory and inhibitory effects on apoptotic signaling. Previous studies by our group demonstrated that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell number, hinders early postimplantation blastocyst development, and increases early-stage blastocyst death. Here, we further investigate the effects of GKB on oocyte maturation, and subsequent pre- and postimplantation development in vitro and in vivo. In our experiments, GKB induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with 1-6 microM GKB during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased placental and fetal weights. Data obtained using an in vivo mouse model further disclosed that consumption of drinking water containing 3-6 microM GKB led to decreased oocyte maturation and in vitro fertilization, as well as early embryo developmental injury, specifically, inhibition of development to the blastocyst stage in vivo. To our knowledge, this is the first study to investigate the impact of GKB on maturation of mouse oocytes, fertilization, and sequential embryonic development.

L-carnitine supplementation reduces oocyte cytoskeleton damage and embryo apoptosis induced by incubation in peritoneal fluid from patients with endometriosis.

OBJECTIVE: To investigate the protective effect of L-carnitine (LC) against deleterious substances present in the peritoneal fluid (PF) of patients with endometriosis, which may affect the oocyte cytoskeleton and embryogenesis. DESIGN: Experimental study. SETTING: Research embryology laboratory at an academic hospital. PATIENT(S): Frozen metaphase II mouse oocytes and embryos. INTERVENTION(S): One hundred metaphase II mouse oocytes were divided into five groups and incubated: PF from endometriosis patients; PF from endometriosis patients + LC; PF from tubal ligation patients (patient control); LC only; and human tubal fluid (HTF) alone. A total of 180 eight-cell mouse embryos were divided into: endometriosis only; tubal ligation only; endometriosis + LC; LC alone; and HTF alone. MAIN OUTCOME MEASURE(S): Protective effect of LC on oocytes and embryos. RESULT(S): Incubation of the oocytes and the embryos with PF from patients with endometriosis statistically significantly damaged the oocyte microtubules and chromosomes and increased embryo apoptosis compared with controls. Incubation with LC (0.6 mg/mL) statistically significantly improved microtubule and chromosome structure and decreased the level of embryo apoptosis. CONCLUSION(S): We propose the use of LC as a supplement in patients with endometriosis, a novel approach that may help improve in vitro fertilization outcome in these patients.

Alteration of mouse oocyte quality after a subchronic exposure to depleted Uranium.

Gametes and embryo tissues are known to represent a sensitive target to environmental toxicants exposure. Oocyte quality can impact subsequent developmental competence, pregnancy course and even adult health. The major health concern from depleted uranium (DU) is mainly centred on its chemotoxic properties as a heavy metal. Little attention was paid to the impact of uranium on female gamete quality. The aim of this research was to evaluate the effect of DU on mouse oocyte quality after 49 days of subchronic contamination in drinking water and to correlate the observed effects with the amount of DU accumulated in organs. Four different DU concentrations were investigated: 0 (control), 10 (DU10), 20 (DU20) and 40 mg L(-1) (DU40). DU did not influence the intensity of ovulation but affected oocyte quality. The proportion of healthy oocytes was reduced by half (P<0.001) from 20 mg L(-1) compared with control group (0.537; 0.497; 0.282 and 0.239 in control, DU10, DU20 and DU40 groups respectively) whereas no accumulation of DU was recorded in the ovaries whatever the dose tested. Abnormal perivitelline space (P<0.001) or absence of the 1st polar body (P<0.001) was identified as the main characteristic of DU impact. In the context of this study, the NOAEL for oocyte quality was determined at 10 mg L(-1) in drinking water (1.9 mg kg(-1)day(-1)). An increase in the dose of contamination over 20 mg L(-1) did not amplify the proportion of oocytes contracting a specific alteration but conducted to a diversification in oocytes abnormalities. Further investigations are necessary to correlate morphologic assessment of female gamete with its developmental competence.

Natural uranium disturbs mouse folliculogenesis in vivo and oocyte meiosis in vitro.

We investigated whether uranium intoxication affects female fertility by assessing its effects on ovarian function and on the oocyte. We treated two groups of female mice for 15 weeks with 5, 50 or 400 mg/L of uranyl nitrate in drinking water. In the first group, mice were euthanized immediately after intoxication. Mice of the second group were paired after intoxication with untreated males. Dams and their female pups were euthanized 3 months after the end of intoxication. We assayed the kidneys, femurs and one ovary per female for U content and collected the other ovary for histology. The number and size of all the ovarian follicles were analyzed. Mice from the first group and female pups had significantly fewer large antral follicles (Ø > 200 microm) than the untreated mice. By contrast, dams in the second group had more secondary and early preantral follicles (Ø 70-110 microm) than untreated mice. However, U had no effect on follicle atresia. We then analyzed the in vitro effects of U on oocyte maturation and fragmentation. GV-oocytes were cultured in the presence of 1mM uranyl acetate and observed for 72 h. Oocyte maturation was slowed down by U during resumption of meiosis and at metaphase II. However, the rhythm and rate of oocyte fragmentation were similar to those of control mice. Our findings demonstrate that U induces changes in folliculogenesis and oocyte maturation in mice and could consequently represent a risk for women who are chronically exposed.