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Our previous research has shown that melatonin (MLT) can reduce cryopreserved ovarian damage in mice. Yet, the molecular mechanism of MLT protection is still unclear. Some studies have shown that melatonin receptor 1 (MT1) is very important for animal reproductive system. To evaluate whether MLT exerts its protective effect on cryopreserved mice ovarian tissue via MT1, we added antagonist of MT1/MT2 (Luzindor) or antagonist of MT2 (4P-PDOT) to the freezing solution, followed by cryopreservation and thawing of ovarian tissue. The levels of total superoxide dismutase (T-SOD), catalase (CAT), nitric oxide (NO) and malondialdehyde (MDA) were detected. Besides, by using RT-PCR and Western blotting, the expression of Bcl-2, Bax and Nrf2/HO-1 signalling pathway-related proteins was detected. These findings demonstrated that compared with the melatonin group, the addition of Luzindor increased apoptosis, NO and MDA activities, decreased CAT and T-SOD activities and inhibited Nrf2/HO-1 signalling pathway. In conclusion, melatonin can play a protective role in cryopreserved ovarian tissue of mice through MT1 receptor.
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Criopreservación , Melatonina , Factor 2 Relacionado con NF-E2 , Ovario , Estrés Oxidativo , Receptor de Melatonina MT1 , Transducción de Señal , Animales , Femenino , Melatonina/farmacología , Estrés Oxidativo/efectos de los fármacos , Ovario/efectos de los fármacos , Receptor de Melatonina MT1/metabolismo , Receptor de Melatonina MT1/genética , Transducción de Señal/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Ratones , Criopreservación/veterinaria , Triptaminas/farmacología , Apoptosis/efectos de los fármacos , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo Oxigenasa (Desciclizante)/genética , Óxido Nítrico/metabolismo , Malondialdehído/metabolismo , Proteínas de la Membrana , Hemo-Oxigenasa 1RESUMEN
The method of immersion optical clearing reduces light scattering in tissues, which improves the use of optical technologies in the practice of clinicians. In this work, we studied the optical and molecular diffusion properties of cat ovarian tissues in the follicular (F-ph) and luteal (L-ph) phases under the influence of glycerol using reflectance spectroscopy in a broad wavelength range from 200 to 800 nm. It was found that the reflectance and transmittance of the ovaries are significantly lower in the range from 200 to 600 nm than for longer wavelengths from 600 to 800 nm, and the efficiency of optical clearing is much lower for the ovaries in the luteal phase compared to the follicular phase. For shorter wavelengths, the following tissue transparency windows were observed: centered at 350 nm and wide (46 ± 5) nm, centered at 500 nm and wide (25 ± 7) nm for the F-ph state and with a center of 500 nm and a width of (21 ± 6) nm for the L-ph state. Using the free diffusion model, Fick's law of molecular diffusion and the Bouguer-Beer-Lambert radiation attenuation law, the glycerol/tissue water diffusion coefficient was estimated as D = (1.9 ± 0.2)10-6 cm2/s for ovaries at F-ph state and D = (2.4 ± 0.2)10-6 cm2/s-in L-ph state, and the time of complete dehydration of ovarian samples, 0.8 mm thick, as 22.3 min in F-ph state and 17.7 min in L-ph state. The ability to determine the phase in which the ovaries are stated, follicular or luteal, is also important in cryopreservation, new reproductive technologies and ovarian implantation.
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Conventional ovarian tissue cryopreservation often destroys the structural, functional, and DNA integrity of the ovarian tissue. How to effectively retain the ultrastructure and subsequent function of ovarian tissue during cryopreservation has long been an issue of concern. Late embryogenesis abundant (LEA) proteins are a class of highly hydrophilic proteins and have been reported to protect various cells from water stress. However, whether LEA proteins exert protective effects on ovarian tissue cryopreservation remains unknown. To investigate the benefit of LEA proteins in ovarian tissue cryopreservation, we purified the recombinant AavLEA1 protein, a member of Group 3 LEA proteins, then cryopreserved the mouse ovaries with this protein by vitrification, and obtained the ovarian follicle structure, cellular proliferation, apoptosis, and GAPDH gene expression of postcryopreservation ovaries. We found that recombinant AavLEA1 protein protected the ovarian follicles from cryoinjury, improved the proliferative ability of follicles, decreased the apoptosis, and promoted the GAPDH gene expression. These results indicated that the LEA protein enhanced the antiapoptosis ability of ovarian cells and retained DNA/RNA integrity against cryoinjury during ovarian tissue vitrification. LEA proteins exert beneficial effects on ovarian tissue cryopreservation, and maybe provide a novel cryoprotective agent for ovarian tissue cryopreservation.
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Criopreservación , Vitrificación , Animales , Criopreservación/métodos , Crioprotectores/química , Crioprotectores/farmacología , Femenino , Ratones , Folículo Ovárico , OvarioRESUMEN
BACKGROUND: Litter size is an important index of mammalian prolificacy and is determined by the ovulation rate. The ovary is a crucial organ for mammalian reproduction and is associated with follicular development, maturation and ovulation. However, prolificacy is influenced by multiple factors, and its molecular regulation in the follicular phase remains unclear. METHODS: Ten female goats with no significant differences in age and weight were randomly selected and divided into either the high-yielding group (n = 5, HF) or the low-yielding group (n = 5, LF). Ovarian tissues were collected from goats in the follicular phase and used to construct mRNA and miRNA sequencing libraries to analyze transcriptomic variation between high- and low-yield Yunshang black goats. Furthermore, integrated analysis of the differentially expressed (DE) miRNA-mRNA pairs was performed based on their correlation. The STRING database was used to construct a PPI network of the DEGs. RT-qPCR was used to validate the results of the predicted miRNA-mRNA pairs. Luciferase analysis and CCK-8 assay were used to detect the function of the miRNA-mRNA pairs and the proliferation of goat granulosa cells (GCs). RESULTS: A total of 43,779 known transcripts, 23,067 novel transcripts, 424 known miRNAs and 656 novel miRNAs were identified by RNA-seq in the ovaries from both groups. Through correlation analysis of the miRNA and mRNA expression profiles, 263 negatively correlated miRNA-mRNA pairs were identified in the LF vs. HF comparison. Annotation analysis of the DE miRNA-mRNA pairs identified targets related to biological processes such as "estrogen receptor binding (GO:0030331)", "oogenesis (GO:0048477)", "ovulation cycle process (GO:0022602)" and "ovarian follicle development (GO:0001541)". Subsequently, five KEGG pathways (oocyte meiosis, progesterone-mediated oocyte maturation, GnRH signaling pathway, Notch signaling pathway and TGF-ß signaling pathway) were identified in the interaction network related to follicular development, and a PPI network was also constructed. In the network, we found that CDK12, FAM91A1, PGS1, SERTM1, SPAG5, SYNE1, TMEM14A, WNT4, and CAMK2G were the key nodes, all of which were targets of the DE miRNAs. The PPI analysis showed that there was a clear interaction among the CAMK2G, SERTM1, TMEM14A, CDK12, SYNE1 and WNT4 genes. In addition, dual luciferase reporter and CCK-8 assays confirmed that miR-1271-3p suppressed the proliferation of GCs by inhibiting the expression of TXLNA. CONCLUSIONS: These results increase the understanding of the molecular mechanisms underlying goat prolificacy. These results also provide a basis for studying interactions between genes and miRNAs, as well as the functions of the pathways in ovarian tissues involved in goat prolificacy in the follicular phase.
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Fase Folicular , MicroARNs , Animales , Femenino , Perfilación de la Expresión Génica , Cabras/genética , MicroARNs/genética , Ovario , ARN Mensajero/genéticaRESUMEN
AIM: To ascertain the actual outcomes of oncofertility care in young women to provide more appropriate care. MATERIALS & METHODS: We analyzed the data of 67 female patients under 43 years of age who underwent oncofertility care between January 2015 and September 2019. RESULTS: There were 28 patients with breast cancer, 19 patients with hematologic cancer and 20 patients with other cancer diagnoses. Breast cancer patients tended to take longer than hematologic cancer patients to initiate oncofertility treatment. Despite undergoing oncofertility care, seven of nine pregnant patients did not choose assisted reproductive technology (ART). CONCLUSION: As spontaneous pregnancies were more common than ART pregnancies in our study, pregnancy by not only ART but also non-ART method is a viable option for young cancer survivors.
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<b>Background and Objective:</b> Female infertility and reproductive problems have increased worldwide. Medical treatment of such conditions has high costs with various side effects. Alternative medicine, essentially herbal plants, has been projecting to improve female infertility and reproductive health. This study was aimed to evaluate the efficacy of single or combined administration of matcha and ashwagandha teas against H<sub>2</sub>O<sub>2</sub>-induced Utero-ovarian oxidative injury and cell death in female rats. <b>Materials and Methods:</b> Fifty adult female rats were used. Ten rats were kept healthy while in others Utero-ovarian oxidative injury was induced by drinking 1% H<sub>2</sub>O<sub>2</sub> water <i>ad libitum</i>. Injured rats were divided into 4 groups (10 rats/each), one group set as injured control and the other 3 groups the doses of supplemented teas were 200 mg kg<sup></sup><sup>1</sup> b.wt. and 100 mg kg<sup></sup><sup>1</sup> b.wt. from each or both teas, respectively. <b>Results:</b> The results displayed that both teas contain active components including flavonoids, polyphenols and possess antioxidant activity. Drinking 1% H<sub>2</sub>O<sub>2</sub> water significantly (p<u><</u>0.01)decreased the estrous cycle time, body, ovary and uterus weights, serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone and estrogen (E2) levels, uterine and ovarian superoxide dismutase (SOD) activity and reduced glutathione (GSH) level while caused a substantial increase (p<u><</u>0.01) in uterine and ovarian malondialdehyde (MDA) level, DNA fragmentation percent, caspase-3 (Casp-3), 8-hydroxydeoxyguanosine (8-OHdG), tumour necrosis factor-α (TNF-α), prostaglandin E2 (PGE2) levels as well as cyclooxygenase-2 (COX-2) activity. Moreover, microscopic observations of uterine and ovarian tissues were consistent with the biochemical results. <b>Conclusion:</b> Oral administration of tested teas improved and ameliorated all the biochemical and microscopic observations by restricting cellular DNA damage and protecting uterine and ovarian tissues from oxidative injury and cell death. The best improvement was observed in the matcha administered group.
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Camellia sinensis/metabolismo , Útero/efectos de los fármacos , Withania/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Ovario/efectos de los fármacos , Ovario/lesiones , Ratas , Resultado del Tratamiento , Útero/lesionesRESUMEN
Oxidative damage is one of the main causes of cryopreservation injury compromising the use of cryopreserved biospecimens. The aim of this study was to evaluate the use of Fourier transform infrared spectroscopy (FTIR) as a non-invasive method to assess changes in biomolecular composition and structure, associated with oxidative stress in isolated biomolecules, acellular heart valve tissues, and ovarian cortex tissues. FTIR spectra of these specimens subjected to various treatments (H2O2- and Fenton-treatment or elevated temperatures) were vector normalized and selected spectral regions were analyzed by principal component analysis (PCA). Control and damaged biomolecules can easily be separated using PCA score plots. Acellular heart valve tissues that were subjected to different levels of oxidative damage formed separate cluster in PCA score plots. In hydrated ovarian tissue, large variation of the principal components was observed. Drying the ovarian tissues samples resulted in improved cluster separation of treatment groups. However, early signs of oxidative damage under mild stress conditions could not be detected by PCA of FTIR spectra. For the ovarian tissue samples, the standardly used nitro blue tetrazolium chloride (NBT) assay was used to monitor the amount of formazan production, reflecting reactive oxygen species (ROS) production at various temperatures. At 37 °C, formazan staining rapidly increased during the first 30 min, and then slowly reached a saturation level, but also at lower temperatures (i.e. 4 °C) formazan production was observed. In summary, we conclude that ATR-FTIR combined with PCA can be used to study oxidative damage in biomolecules as well as in tissues. In tissues, however, sample heterogeneity makes it difficult to detect early signs of oxidative damage.
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Peróxido de Hidrógeno , Estrés Oxidativo , Análisis de Componente Principal , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Cryopreservation of ovarian tissues (OTs) has become the most effective way to preserve the fertility of female cancer patients. However, cryopreservation of OTs is still relatively at an experimental stage. The aim of study is to examine the effect of melatonin (MTL) on cryopreserved-thawed OTs. Fragments of OTs were cryopreserved in medium containing different concentrations (0 mM, 0.001 mM, 0.01 mM, 0.1 mM and 1 mM) of MLT. The endogenous enzymes (GSH-PX, GSH, SOD, CAT and T-AOC), MDA and ROS levels were all evaluated after cryopreservation. Our results showed that the 0.1 mM of MLT significantly improved the survival and diameter of follicles (P < 0.001). Meanwhile, the antioxidant enzymes activities (including GSH-PX, GSH, SOD, CAT and T-AOC) were enhanced and MDA content were significantly decreased in 0.1 mM of MLT group compared to other groups (P < 0.001). Additionally, compared to the control group, MTL of 0.1 mM resulted in a significantly lower ROS level. In conclusion, MLT protects the quality of cryopreserved OTs by decreasing oxidative stress level and the optimal concentration is 0.1 mM.
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Antioxidantes , Melatonina , Animales , Antioxidantes/farmacología , Criopreservación/métodos , Femenino , Glutatión Peroxidasa/metabolismo , Humanos , Malondialdehído , Melatonina/farmacología , Ratones , Estrés OxidativoRESUMEN
Growth retardation and gonadal dysgenesis are two of the most important clinical manifestations of Turner syndrome (TS). As premature ovarian failure generally occurs early in life in women with TS, these patients should be counseled and evaluated as early as possible for discussion of optimal and individualized fertility preservation strategies. Infertility seriously affects the quality of life of women with TS. For those who have ovarian reserve, the theoretical options for future fertility in TS patients include cryopreservation of oocytes, ovarian tissues, and embryos. For those who have already lost their ovarian reserve, oocyte or embryo donation, gestational surrogacy, and adoption are strategies that allow fulfillment of desire for parenting. This review describes the etiologies of infertility and reviews the fertility preservation strategies for women with TS.
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Polyploidization can significantly alter the size of animal gametes. Autotetraploid fish (RRRR, 4nRR = 200) (4nRR) possessing four sets of chromosomes were derived from whole-genome duplication in red crucian carp (RR, 2n = 100) (RCC). The diploid eggs of the 4nRR fish were significantly larger than the eggs of RCC. To explore the differences between the ovaries of these two ploidies of fishes at the molecular level, we compared the ovary transcriptome profiles of 4nRR fish and RCC and identified differentially expressed genes (DEGs). A total of 19,015 unigenes were differentially expressed between 4nRR fish and RCC, including 12,591 upregulated and 6,424 downregulated unigenes in 4nRR fish. Functional analyses revealed that eight genes (CDKL1, AHCY, ARHGEF3, TGFß, WNT11, CYP27A, GDF7, and CKB) were involved in the regulation of cell proliferation, cell division, gene transcription, ovary development and energy metabolism, suggesting that these eight genes were related to egg size in 4nRR fish and RCC. We validated the expression levels of these eight DEGs in 4nRR fish and RCC using quantitative PCR. The study results provided insights into the regulatory mechanisms underlying the differences in crucian carp egg sizes.
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BACKGROUND: To reduce young female fertility loss, the in-vitro culture of cryopreserved ovarian cortical tissues (OCTs) is considered an effective approach without delaying treatment and undergoing stimulation medicine. However, ischemic damage and follicular loss during the in-vitro culture of OCTs are major technical challenges. Human umbilical cord stem cells (HUMSCs) and their conditioned medium (HUMSC-CM) have been considered to be potential resources for regeneration medicine because they secrete cytokines and enhance cell survival and function. The aim of this study was to determine whether HUMSC-CM improves the development of frozen-thawed in-vitro cultured ovarian tissues compared with a serum-free culture medium (SF-CM). METHODS: The thawed OCTs (n = 68) were cultivated in HUMSC-CM and SF-CM in vitro for 8 days, and the ovarian tissues were processed and analyzed by a classical histological evaluation. The microvessel density (MVD) and apotosis detection during in-vitro culture of OCTs were also performed. RESULTS: A significant difference in the rate of morphologically normal primordial follicles in the HUMSC-CM group was observed compared to that in the SF-CM group (group C) from days 2 to 4 (day 2: group B 58.0 ± 2.45% vs group C 32.0 ± 5.83%, p = 0.002; day 3: group B 55.5 ± 4.20% vs group C 21.0 ± 9.80%, p = 0.048; day 4: group B 52.0 ± 4.08% vs group C 21.5 ± 8.19%, p = 0.019). The microvessel density (MVD) detection showed a time-dependent increase and peaked on day 4. There was a significant difference between groups B (49.33 ± 0.58) and C (24.33 ± 3.79) (p = 0.036). The percentage of apoptotic follicles in group B was lower than that in group C on day 1 (13.75 ± 2.50% vs 27.0 ± 10.10%, p = 0.003), day 5 (11.75 ± 1.50% vs 51.0 ± 10.5%, p = 0.019) and day 7 (15.0 ± 5.10% vs 46.5 ± 21.75%, p = 0.018). CONCLUSIONS: These data have provided the first experimental evidence of the effect of HUMSC-CM on frozen-thawed OCTs in vitro. The results showed that the HUMSC-CM group provided a better protecting effect on the in-vitro culture of the cryopreserved OCTs compared to the SF-CM group.
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Criopreservación , Medios de Cultivo Condicionados/farmacología , Medio de Cultivo Libre de Suero/farmacología , Ovario/metabolismo , Células Madre/metabolismo , Cordón Umbilical/metabolismo , Adulto , Femenino , Humanos , Técnicas de Cultivo de Órganos/métodos , Ovario/citología , Células Madre/citología , Cordón Umbilical/citologíaRESUMEN
Corticosteroids play positive or negative role in the reproductive mechanisms of many fish species but the physiological contexts relating to such biphasic actions are not well defined. In the present study we investigated to what extent corticosteroids (cortisol-Co, 11-deoxycorticosterone-DOC) hormones may interfere with the steroidogenic capacity of Eurasian perch ovarian tissues, and we tested whether the negative effects of corticosteroids may be mitigated by potential stimulating endocrine factors, namely insulin-like growth factor-1 (IGF), human chorionic gonadotropin (HCG) or thyroid hormones (Triidothyronine-T3, thyroxine-T4). Ovarian tissues from six maturing fish at late vitellogenesis developmental stage (LVO) or at the start of the final meiotic oocyte maturation (FMO) were incubated during 6h in Cortland medium containing various endocrine compounds. Both corticosteroids drastically suppressed aromatase activity (AA) and sex-steroid production, namely 17-ß estradiol (E2), 17α-20ß-dihydroxy-4-pregnen-3-one (DHP) and testosterone (T). HCG significantly prevented the suppression of both AA and sex-steroid production by low and high cortisol doses, but a lesser AA protection was observed in the case of DOC. The protection of DHP and T productions by HCG from the negative effects by the two corticosteroids was higher at FMO than at LVO stage. IGF or thyroid hormone treatments were lesser effective or ineffective in mitigating the suppression of AA or sex-steroid production by cortisol. The results suggest that an increase in cortisol or DOC such as after mild or high stress intensity may inhibit drastically the ovarian steroidogenic capacity whatever the final oocyte maturation stage in percid fish by hampering AA and sex-steroid production. That inhibition may be partly mitigated by gonadotropins but not IGF nor thyroid hormones, especially at final meiotic oocyte maturation stage.
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Desoxicorticosterona/farmacología , Hormonas Esteroides Gonadales/metabolismo , Hidrocortisona/farmacología , Percas/fisiología , Animales , Aromatasa/genética , Aromatasa/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hormonas Esteroides Gonadales/genética , Humanos , Oocitos/crecimiento & desarrollo , Ovario/efectos de los fármacos , Ovario/metabolismo , Receptor IGF Tipo 1/farmacología , Tiroxina/farmacología , Técnicas de Cultivo de Tejidos , Triyodotironina/farmacologíaRESUMEN
RNA sequencing and short-read assembly was utilized to produce a transcriptome of ovarian tissues from three-year-old diploid and tetraploid loaches (Misgurnus anguillicaudatus). A total of 28,369 unigenes were obtained, comprising 10,546 unigenes with length longer than 1000 bp. More than 73% of the unigenes were annotated through sequence comparison with databases. The RNA-seq data revealed that 2253 genes were differentially expressed between diploid and tetraploid loaches, including 1263 up-regulated and 990 down-regulated genes in tetraploid loach. Some differentially expressed genes, such as vitellogenin (Vtg), gonadotropin releasing hormone receptor type A (GnRHRA), steroidogenic acute regulatory protein (StAR), mitogen-activated protein kinase 14a (MAPK14a), ATP synthase subunit alpha (atp5a), and synaptonemal complex protein 1 (Scp1), were involved in regulation of cell proliferation, division, gene transcription, ovarian development and energy metabolism, suggesting that these genes were related to egg diameter of the loach. Results of transcriptome profiling here were validated using real time quantitative PCR in ten selected genes. The present study provided insights into the transcriptome profile of ovarian tissues from diploid and tetraploid loaches Misgurnus anguillicaudatus, which was made available to the research community for functional genomics, comparative genomics, polyploidy evolution and molecular breeding of this loach and other related species.
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Cipriniformes/genética , Ovario/metabolismo , Transcriptoma , Animales , Cipriniformes/metabolismo , Diploidia , Etiquetas de Secuencia Expresada , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , ARN/química , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ARN , TetraploidíaRESUMEN
The aim of the present study was to establish human ovarian stroma within the mouse subcutaneously, in order for the resulting stroma to serve as a useful preclinical tool to study the progression of human ovarian cancer in a humanized ovarian microenvironment. Normal human ovarian tissues were subcutaneously implanted into severe combined immunodeficient (SCID) mice and then the implants were identified by immunohistochemistry. The implants became vascularized and retained their original morphology for about 4 weeks following implantation. Immunohistochemical staining for cytokeratin-7 confirmed the ovarian origin of the epithelial cells. CD34 staining demonstrated human-derived vessels. Positive estrogen receptor and partially-positive progesterone receptor staining indicated the estrogen and progesterone dependence of the implants. Only vascular pericytes expressed α-smooth muscle actin, indicating the normal ovarian origin of the xenografts. Human ovarian tissue successfully survived in SCID mice and retained its original properties. This humanized mouse model may be used as preclinical tool to investigate ovarian cancer.
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The environmental persistence and bioaccumulation of herbicide atrazine may pose a significant threat to human health. In this experiment, Wistar rats were treated by 5, 25 and 125 mg·kg(-1) atrazine respectively for 28 days, and the oxidative stress responses as well as the activations of Nrf2 signaling pathway in ovarian tissues induced by atrazine were observed. The results showed that after be treated by atrazine, the proportion of atretic follicles in the rat ovary were increased, the contents of NO and MDA in the tissue homogenates were increased, the over-expressed Nrf2 transferred into the nuclei and played an antioxidant role by up-regulated the expression of II phase detoxifying enzymes such as HO1 and NQO1 and the expression of antioxidant enzymes such as CAT, SOD and GSH-PX.