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Objective: To demonstrate the changes of ovarian aging markers across the Stages of Reproductive Aging Workshop (STRAW) stages and modify it with subclassification of mid reproductive age stage (MR). Design: Healthy females were classified according to the STRAW system. Serum basal FSH, LH, E2, and anti-Müllerian hormone (AMH) were detected, FSH/LH ratio calculated, and antral follicle counts (AFCs) determined in follicular phase. Results: Progression through the whole STRAW stages under MR stage subdivided is associated with elevations in FSH, LH, FSH/LH ratio and decreases in E2, AMH and AFCs ( p < 0.001). Both serum AMH and AFCs decreased early (after 25 years) and significantly ( p < 0.01) with chronological age in MR stage. 0.982 ng/ml AMH and 3 antral follicles (low level of MR 25-30 years) were set as cutoffs to distinguish MR stage into early mid reproductive age (EMR) and late mid reproductive age (LMR) stages. The women in EMR stage compared with LMR could retrieve more oocytes in IVF treatment ( p < 0.05) and has a higher pregnancy chance (57.9%) though not significant. Conclusion(s): The early and marked fall in serum AMH levels and AFCs suggest fine markers to further categorize and define the MR stage, demonstrating disparate reproductive aging period with reduced ovarian reserve in young age across the STRAW stages. [ABSTRACT FROM AUTHOR]

Abstract: c-FLIP inhibits caspase-8 activation and cell apoptosis mediated by death receptors. The present study aims at determining the effects of c-FLIP targeted vector-based short hairpin RNA (shRNA) on cell growth and evaluating its modulation of responsiveness to drugs and radiotherapy in cervical adenocarcinoma Hela cells. cFLIP expression of the cells transfected with shRNA against c-FLIP was significantly down-regulated after 72h. c-FLIP silencing markedly suppressed cell proliferation and increased cell apoptosis. The activation of caspase-8 and caspase-3 was induced with shRNA targeting cFLIP with the passage of time after transfection. Furthermore, Vector-based shRNA against c-FLIP subsequently increased the sensitivity to cisplatin, iritican and Co60 radiotherapy by about 4- to 6-folds in Hela cells. Our data suggest that vector-based shRNA effectively inhibited c-FLIP expression, enhanced the expression level of caspase-8 and caspase-3 to induce cell apoptosis, probably with the higher efficacy in combination therapies with conventional chemotherapy and radiotherapy in cervical adenocarcinoma. [Copyright &y& Elsevier]

Our previous study demonstrated that ovarian wild-type P53-induced phosphatase 1 (WIP1) expression decreased with age. We hypothesized that WIP1 activity was related to ovarian aging. The role of WIP1 in regulating ovarian aging and its mechanisms remain to be elucidated. Adult female mice with or without WIP1 inhibitor (GSK2830371) treatment were divided into three groups (Veh, GSK-7.5, GSK-15) to evaluate the effect of WIP1 on ovarian endocrine and reproductive function and the ovarian reserve. In vitro follicle culture and primary granulosa cell culture were applied to explore the mechanisms of WIP1 in regulating follicular development. This study revealed that WIP1 expression in atretic follicle granulosa cells is significantly lower than that in healthy follicles. Inhibiting WIP1 phosphatase activity in mice induced irregular estrous cycles, caused fertility declines, and decreased the ovarian reserve through triggering excessive follicular atresia and primordial follicle activation. Primordial follicle depletion was accelerated via PI3K-AKT-rpS6 signaling pathway activation. In vitro follicle culture experiments revealed that inhibiting WIP1 activity impaired follicular development and oocyte quality. In vitro granulosa cell experiments further indicated that downregulating WIP1 expression promoted granulosa cell death via WIP1-p53-BAX signaling pathway-mediated apoptosis. These findings suggest that appropriate WIP1 expression is essential for healthy follicular development, and decreased WIP1 expression accelerates ovarian aging by promoting follicular atresia and primordial follicle activation. [ABSTRACT FROM AUTHOR]

What role does wild-type p53-induced phosphatase 1 (WIP1) play in the regulation of primordial follicle development? WIP1 expression was detected in the ovaries of mice of different ages by western blotting and immunohistochemical staining. Three-day-old neonatal mouse ovaries were cultured in vitro with or without the WIP1 inhibitor GSK2830371 (10 μM) for 4 days. Ovarian morphology, follicle growth and follicle classification were analysed and the PI3K–AKT–mTOR signal pathway and the WIP1–p53-related mitochondrial apoptosis pathway evaluated. WIP1 expression was downregulated with age. Primordial follicles were significantly decreased in the GSK2830371-treated group, without a significant increase in growing follicles. The ratio of growing follicles to primordial follicles was not significantly different between the control and GSK2830371 groups, and no significant variation was observed in the PI3K–AKT–mTOR signal pathway. The inhibition of WIP1 phosphatase accelerated primordial follicle atresia by activating the p53–BAX–caspase-3 pathway. These findings reveal that WIP1 participates in regulating primordial follicle development and that inhibiting WIP1 phosphatase leads to massive primordial follicle loss via interaction with the p53–BAX–caspase-3 pathway. This might also provide valuable information for understanding decreased ovarian reserve during ovarian ageing. [ABSTRACT FROM AUTHOR]

As a fundamental aging mechanism, cellular senescence causes chronic inflammation via the senescence‐associated secretory phenotype (SASP). Theca‐interstitial cells are an essential but little‐studied component of follicle development in the ovarian microenvironment. In the present study, we observed significant cellular senescence in theca‐interstitial cells and secretion of chemokine (C‐C motif) ligand 5 (CCL5) by these cells during aging. Furthermore, we aimed to investigate whether and how senescence‐associated secretory phenotype (SASP)‐associated CCL5 may be involved in follicle development. Increased levels of CCL5 in the microenvironment of follicles attenuated preantral follicle growth, survival, and estradiol secretion. Oocyte maturation and the expression of zona pellucida 3 and differentiation factor 9 (GDF9) were also inhibited by CCL5. Granulosa cell apoptosis in follicles was promoted by CCL5, accompanied by the phosphorylation of nuclear factor‐κB by CCL5 and inhibition of the PI3K/AKT pathway. These results suggest that SASP‐associated CCL5 produced by senescent theca‐interstitial cells may impair follicle development and maturation during ovarian aging by promoting granulosa cell apoptosis. [ABSTRACT FROM AUTHOR]

There are about 1-2 million follicles presented in the ovary at birth, while only around 1000 primordial follicles are left at menopause. The ovarian function also decreases in parallel with aging. Folliculogenesis is vital for ovarian function, no matter the synthesis of female hormones or ovulation, yet the mechanisms for its changing with increasing age are not fully understood. Early follicle growth up to the large preantral stage is independent of gonadotropins in rodents and relies on intraovarian factors. To further understand the age-related molecular changes in the process of folliculogenesis, we performed microarray gene expression profile analysis using total RNA extracted from young (9 weeks old) and old (32 weeks old) mouse ovarian secondary follicles. The results of our current microarray study revealed that there were 371 (≥2-fold, q-value ≤0.05) genes differentially expressed in which 174 genes were upregulated and 197 genes were downregulated in old mouse ovarian secondary follicles compared to young mouse ovarian secondary follicles. The gene ontology and KEGG pathway analysis of differentially expressed genes uncovered critical biological functions such as immune system process, aging, transcription, DNA replication, DNA repair, protein stabilization, and apoptotic process were affected in the process of aging. The considerable changes in gene expression profile may have an adverse influence on follicle quality and folliculogenesis. Our study provided information on the processes that may contribute to age-related decline in ovarian function. [ABSTRACT FROM AUTHOR]

Mammalian oocytes go through a long and complex developmental process, while acquiring the competencies that are required for fertilization and embryogenesis. Recent studies revealed that the communication between oocytes and granulosa cells (GCs) is a critical process for female follicle development. In the current study, we aimed to study whether and how semaphorin 6C (Sema6c) regulated the cell junctions between oocytes and GCs in mice preantral follicles. The attenuation of SEMA6C expression by siRNA decreased the cell–cell junctions and accelerated follicle atresia in vitro. PI3K‐AKT pathway was activated when SEMA6C expression was downregulated. And the LY294002, a PI3K inhibitor, could reverse the effect of low SEMA6C expression on cell junctions in preantral follicles. Our findings revealed that Sema6c was involved in follicle development, and the suppression of SEMA6C led to cell junction defection by activating the PI3K/AKT pathway, which might also provide valuable information for understanding premature ovarian failure and ovarian aging. Mammalian oocytes go through a long and complex developmental process, while acquiring the competencies that are required for fertilization and embryogenesis.Our findings revealed that Sema6c was involved in follicle development, and the suppression of SEMA6C led to cell junction defection by activating the PI3K/AKT pathway, which might also provide valuable information for understanding premature ovarian failure and ovarian aging. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) are a unique class of endogenous RNAs which could be used as potential diagnostic and prognostic biomarkers of many diseases. Our study aimed to investigate circRNA profiles in human granulosa cells (GCs) during maternal aging and to uncover age-related circRNA variations that potentially reflect decreased oocyte competence. CircRNAs in GCs from in vitro fertilization (IVF) patients with young age (YA, ≤ 30 years) and advanced age (AA, ≥ 38 years) were profiled by microarray, and validated in 20 paired samples. The correlation between circRNAs expression and clinical characteristics was analyzed in additional 80 samples. Chip-based analysis revealed 46 up-regulated and 11 down-regulated circRNAs in AA samples (fold change > 2.0). Specifically, circRNA_103829, circRNA_103827 and circRNA_104816 were validated to be up-regulated, while circRNA_101889 was down-regulated in AA samples. After adjustment for gonadotropin treatment, only circRNA_103827 and circRNA_104816 levels were positively associated with maternal age (partial r = 0.332, P = 0.045; partial r = 0.473, P = 0.003; respectively). Moreover, circRNA_103827 and circRNA_104816 expressions in GCs were negatively correlated with the number of top quality embryos (r = -0.235, P = 0.036; r = -0.221, P = 0.049; respectively). Receiver operating characteristic (ROC) curve analysis indicated that the performance of circRNA_103827 for live birth prediction reached 0.698 [0.570–0.825], with 77.2% sensitivity and 60.9% specificity (P = 0.006), and that of circRNA_104816 was 0.645 [0.507–0.783] (P = 0.043). Bioinformatics analysis revealed that both circRNAs were potentially involved in glucose metabolism, mitotic cell cycle, and ovarian steroidogenesis. Therefore, age-related up-regulation of circRNA_103827 and circRNA_104816 might be potential indicators of compromised follicular micro-environment which could be used to predict IVF prognosis, and improve female infertility management. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Kuntai capsule, a traditional Chinese medicine, has been widely used for the clinical treatment of menopausal syndrome. However, its mechanisms remain poorly understood. Considering that aging ovaries are the primary cause of menopause, this study was designed to investigate the effects and mechanisms of Kuntai capsule on ovarian function in a novel mice model with accelerated aging ovaries. Materials and methods Seventy-five female C57BL/6 mice were chosen for this study. Fifteen of the mice were separated into the normal control group (NC). The remaining sixty were used to establish the novel accelerated aging ovary model by superovulation and oxidative stress and then by randomly dividing the mice into four equal groups. One group was considered the model group (Mod). The other three groups were treated with low (0.4 g/kg), middle (0.8 g/kg) and high (1.6 g/kg) doses of Kuntai capsule intragastrically every day for 4 weeks. During the treatment, the body weight and fur condition of all mice were recorded. All the mice were forced to swim to record their exhaustive swimming time (EST), which measures their strength. Mice were then sacrificed for sampling. Ovarian reserve was evaluated using follicle counts and AMH expression. Ovarian function was evaluated using estrous cycle, sex hormone level and litter experiments. Ovarian follicles were categorized and counted to estimate ovarian reserve, and ovarian histologic sections were stained for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) to detect apoptotic cells. The ultrastructure of ovarian cells was observed using transmission electron microscopy. Western blotting was used to measure expression of Bax, Bcl2, AMH and SOD2 protein. Results Compared with the NC GROUP, the Mod group clearly displayed worse fur condition and ovarian function. These situations showed some improvement after Kuntai capsule treatment. Specifically, the fur condition and the EST of the Kuntai capsule groups were superior to the fur condition and EST of the Mod group. In cases of damaged ovarian function, Kuntai capsule can regulate the estrous cycles, increase hormone secretion and fertility and significantly decrease atretic follicles. The transmission electron microscopy results revealed that Kuntai capsule rescued the ovarian ultrastructure of mice. TUNEL staining confirmed that the apoptotic cells were reduced after Kuntai capsule treatment. Western blotting revealed that Kuntai capsule can increase AMH, SOD2, and Bcl2 protein expression and decrease Bax expression. Conclusions Kuntai capsule may improve damaged ovarian function, which may be related to its antioxidant and anti-apoptosis effects. [ABSTRACT FROM AUTHOR]

SIRT1 has many important molecular functions in aging, and the estrogen receptors ( ERs) have a vasculoprotective effect, although the detailed mechanism for the roles of SIRT1 and ERs in vascular aging remains unclear. We found that ERβ expression in the endothelium was reduced in aging mice, and the expression of ERα and SIRT1 did not change, while SIRT1 activity declined. Further investigation showed that the ERβ expression was regulated by SIRT1 through complexes of SIRT1- PPARγ/ RXR-p300 that bind to a PPRE ( PPAR response element) site on the ERβ promoter, and the declined SIRT1 function in aging mice was due to compromised phosphorylation at S154. A single-mutant SIRT1-C152(D) restored the reduced ERβ expression in the endothelium with minimized reactive oxygen species generation and DNA damage and increased mitochondrial function and fatty acid metabolism. In high-fat diet aging mice, the endothelium-specific delivery of ERβ or SIRT1-C152(D) on the vascular wall reduced the circulating lipids with ameliorated vascular damage, including the restored vessel tension and blood pressure. We conclude that SIRT1-mediated ERβ suppression in the endothelium contributes to vascular aging, and the modulation of SIRT1 phosphorylation through a single-mutant SIRT1-C152(D) restores this effect. [ABSTRACT FROM AUTHOR]

Ovarian aging is a long-term and complex process associated with a decrease in follicular quantity and quality. The damaging effects of reactive oxygen species (ROS) in ovarian aging and ovarian aging-associated disorders have received relatively little attention. Thus, we assessed if the oxidative stress induced by long-term (defined by the Environmental Protection Agency as at least 30 days in duration) moderate ozone inhalation reduced ovarian reserves, decreased ovarian function and induced ovarian aging-associated disorders. The expression of oxidative stress markers and antioxidant enzymes was used to determine the degree of oxidative stress. Ultrastructural changes in ovarian cells were examined via electron microscopy. The ovarian reserve was assessed by measuring multiple parameters, such as the size of the primordial follicle pool and anti-Müllerian hormone (AMH) expression. The estrous cycle, hormone levels and fertility status were investigated to assess ovarian function. To investigate ovarian aging-associated disorders, we utilized bone density and cardiovascular ultrasonography in mice. The levels of oxidized metabolites, such as 8-hydroxy-2´-deoxyguanosine (8-OHdG), 4-hydroxynonenal (4-HNE) and nitrotyrosine (NTY), significantly increased in ovarian cells in response to increased oxidative stress. The ultrastructural analysis indicated that lipid droplet formation and the proportion of mitochondria with damaged membranes in granulosa cells were markedly increased in ozone-exposed mice when compared with the control group. Ozone exposure did not change the size of the primordial follicle pool or anti-Müllerian hormone (AMH) expression. The estrogen concentration remained normal; however, progesterone and testosterone levels decreased. The mice exposed to ozone inhalation exhibited a substantial decrease in fertility and fecundity. No differences were revealed by the bone density or cardiovascular ultrasounds. These findings suggest that the decreased female reproductive function caused by long-term moderate oxidative damage may be due to a decrease in follicle quality and progesterone production. [ABSTRACT FROM AUTHOR]

Sirtuins, a family of nicotinamide adenine dinucleotide (NAD)-dependent deacetylases that play diverse roles in regulating metabolism, cell proliferation, and genome stability, have been implicated in mammalian aging and age-related diseases, including cancers, metabolic disorders, and neurodegenerative diseases. Ovarian aging is thought to be characterized by a gradual decrease in both the number of follicles and the quality of oocytes. Ovarian reserve is indicated by the number of primordial follicles. In this study, ovarian reserve was assessed in mice of different ages and mice subjected to caloric restriction (CR) and chemotherapy (2 commonly used models for ovarian aging research) by counting primordial follicles and determining the expression levels of SIRT1, SIRT3, and SIRT6 to explore the relationship between ovarian function and sirtuin expression. A gradual decline in the number of follicles (especially primordial follicles) was observed in aging mice and mice subjected to chemotherapy. Histological analysis showed that CR mice displayed a significantly greater number of primordial follicles and less atretic follicles. Western blot analysis indicated that expression levels of SIRT1, SIRT3, and SIRT6 were significantly decreased in the ovaries of aged mice and mice treated with chemotherapy, but increased in CR mice. SIRT1, SIRT3, and SIRT6 all showed a significantly positive correlation with the numbers of primordial follicles (r 2 = 0.6399, P < 0.0001; r 2 = 0.5445, P < 0.001; and r 2 = 0.4956, P < 0.0001, respectively). These results indicate that SIRT1, SIRT3 and SIRT6 are closely related to ovarian reserve, and suggest that these sirtuins may be markers of ovarian aging. [ABSTRACT FROM AUTHOR]

Female germline stem cells (FGSCs) or oogonial stem cells (OSCs) have the capacity to generate newborn oocytes and thus open a new door to fight ovarian aging and female infertility. However, the production and identification of OSCs are difficult for investigators. Rare amount of these cells in the ovary results in the failure of the acquisition of OSCs. Furthermore, the oocyte formation by OSCs in vivo was usually confirmed using tissue sections by immunofluorescence or immunohistochemistry in previous studies. STO or MEF feeder cells are derived from mouse, not human. In our study, we modified the protocol. The cells were digested from ovaries and cultured for 2-3 days and then were purified by magnetic-activated cell sorting (MACS). The ovaries and fetus of mice injected with EGFP-positive OSCs were prepared and put on the slides to directly visualize oocyte and progeny formation under microscope. Additionally, the human umbilical cord mesenchymal stem cells (hUC-MSCs) were also used as feeder cells to support the proliferation of OSCs. The results showed that all the modified procedures can significantly improve and facilitate the generation and characterization of OSCs, and hUC-MSCs as feeder will be useful for isolation and proliferation of human OSCs avoiding contamination from mouse. [ABSTRACT FROM AUTHOR]

Female germline stem cells (FGSCs) or oogonial stem cells (OSCs) have the capacity to generate newborn oocytes and thus open a new door to fight ovarian aging and female infertility. However, the production and identification of OSCs are difficult for investigators. Rare amount of these cells in the ovary results in the failure of the acquisition of OSCs. Furthermore, the oocyte formation by OSCs in vivo was usually confirmed using tissue sections by immunofluorescence or immunohistochemistry in previous studies. STO or MEF feeder cells are derived from mouse, not human. In our study, we modified the protocol. The cells were digested from ovaries and cultured for 2-3 days and then were purified by magnetic-activated cell sorting (MACS). The ovaries and fetus of mice injected with EGFP-positive OSCs were prepared and put on the slides to directly visualize oocyte and progeny formation under microscope. Additionally, the human umbilical cord mesenchymal stem cells (hUC-MSCs) were also used as feeder cells to support the proliferation of OSCs. The results showed that all the modified procedures can significantly improve and facilitate the generation and characterization of OSCs, and hUC-MSCs as feeder will be useful for isolation and proliferation of human OSCs avoiding contamination from mouse. [ABSTRACT FROM AUTHOR]

Early menopause and infertility often occur in female cancer patients after chemotherapy (CTx). For these patients, oocyte/embryo cryopreservation or ovarian tissue cryopreservation is the current modality for fertility preservation. However, the above methods are limited in the long-term protection of ovarian function, especially for fertility preservation (very few females with cancer have achieved pregnancy with cryopreserved ovarian tissue or eggs until now). In addition, the above methods are subject to their scope (females with no husband or prepubertal females with no mature oocytes). Thus, many females who suffer from cancers would not adopt the above methods pre- and post-CTx due to their uncertainty, safety and cost-effectiveness. Therefore, millions of women have achieved long-term survival after thorough CTx treatment and have desired to rescue their ovarian function and fertility with economic, durable and reliable methods. Recently, some studies showed that mice with infertility caused by CTx can produce normal offspring through intraovarian injection of exogenous female germline stem cells (FGSCs). Though exogenous FGSC can be derived from mice without immune rejection in the same strain, it is difficult to obtain human female germline stem cells (hFGSCs), and immune rejection could occur between different individuals. In this study, infertility in mice was caused by CTx, and the ability of FGSCs to restore ovarian function or even produce offspring was assessed. We had successfully isolated and purified the FGSCs from adult female mice two weeks after CTx. After infection with GFP-carrying virus, the FGSCs were transplanted into ovaries of mice with infertility caused by CTx. Finally, ovarian function was restored and the recipients produced offspring long-term. These findings showed that mice with CTx possessed FGSCs, restoring ovarian function and avoiding immune rejection from exogenous germline stem cells. [ABSTRACT FROM AUTHOR]

Theca-interstitial cells (TICs) and granulosa cells (GCs) are important components of follicles that support follicle development and hormone secretion, and are considered to be important cell models for basic research. However, no method currently exists for simultaneously isolating TICs and GCs from a single ovary of the immature mouse. Here, we sought to develop such a protocol using mechanical dissection combined with brief collagenase-DNase digestion. Morphological characteristics and molecular markers were detected to identify TICs and GCs. In isolated TICs, cholesterol side chain cleavage cytochrome P450 (P450scc) was expressed abundantly, but anti-Mullerian hormone (AMH) was expressed only at very low levels. This expression profile was reversed in GCs. In addition, TICs secreted large amounts of testosterone (T) and minimal amounts of estradiol (E2), while the converse was found in GCs. T concentrations rose gradually in TIC culture media as the concentration of added luteinizing hormone (LH) was increased. In GCs, E2 secretion increased as the follicle-stimulating hormone (FSH) concentration increased. Thus, mechanical dissection combined with collagenase-DNase digestion is a simple, effective and reproducible method for obtaining large numbers of highly purified and hormonally stimulated TICs and GCs from one ovary. [ABSTRACT FROM AUTHOR]

Background: Death-associated protein kinase1 (DAPK1) is an important tumor suppressor gene. DNA methylation can inactivate genes, which has often been observed in the carcinogenesis of cervical cancer. During the past several decades, many studies have explored the association between DAPK1 promoter methylation and cervical cancer. However, many studies were limited by the small samples size and the findings were inconsistent among them. Thus, we conducted a meta-analysis to assess the association between DAPK1 promoter methylation and cervical cancer. Methods: We systematically searched eligible studies in the PubMed, Web of Science, EMBASE and CNKI databases. Using meta-regression, subgroup analysis and sensitivity analysis, we explored the potential sources of heterogeneity. The odds ratio (OR) and 95% confidence interval (95% CI) were calculated by Meta-Analysis in R. Results: A total of 15 studies from 2001 to 2012, comprising 818 tumor tissues samples and 671 normal tissues samples, were analyzed in this meta-analysis. The frequencies of DAPK1 promoter methylation ranged from 30.0% to 78.6% (median, 59.3%) in cervical cancer tissue and 0.0% to 46.7% (median, 7.8%) in normal cervical tissue. The pooled OR was 19.66 (95%CI = 8.72–44.31) with the random effects model, and heterogeneity was found through the sensitivity analysis. The I2 = 60% (P = 0.002) decreased to I2 = 29.2% (P = 0.144) when one heterogeneous study was excluded, and the pooled OR increased to 21.80 (95%CI = 13.44–35.36) with the fixed effects model. Conclusion: The results suggested a strong association between DAPK1 promoter methylation and cervical cancer. This study also indicated that DAPK1 promoter methylation may be a biomarker during cervical carcinogenesis that might serve as an early indication of cervical cancer. [ABSTRACT FROM AUTHOR]

Mouse models have been widely utilized to elucidate the basic principles and regulatory mechanisms of primordial follicle activation. Outside their natural environment, the growth of follicles might be affected by unknown factors in vitro and the elimination of regulation in vivo. Currently, in vitro culture and transplantation of ovaries under the kidney capsule are two commonly used incubation methods. However, the limited number of studies that have been published compare various incubation systems and reveal differences between ovaries that are incubated and grown in vivo. We compare the number of primordial, primary and secondary follicles in cultured, transplanted and in-vivo-grown ovaries. We investigate the expression levels of four genes, including zona pellucida 3 (ZP3), growth and differentiation factor-9 (GDF-9), proliferating cell nuclear antigen (PCNA) and anti-Müllerian hormone (AMH). Our results suggest that in vitro culture accelerates follicle activation, delays the transition from primary to secondary follicles and affects the expression patterns of ZP3, GDF-9, PCNA and AMH. A larger number of secondary follicles in ovaries cultured in alpha-minimal essential medium (α-MEM) had intact zona pellucida compared with those grown in Dulbecco's modified Eagle medium containing Ham's F-12 nutrient mixture (D/F12), suggesting that α-MEM is a better basal medium. The transplanted ovaries demonstrated the most similar characteristics to the in-vivo-grown ovaries, indicating that transplantation provided an optimal environment for ovarian incubation. This study has thus established the similarities and differences between in-vivo-grown and incubated ovaries, demonstrated that transplantation can mostly mimic the environment of ovarian growth in vivo and determined the optimal basal culture medium between α-MEM and D/F12. [ABSTRACT FROM AUTHOR]

Abstract: Recent studies have shown that oncolytic adenovirus specifically targeted tumor cells while sparing normal cells. Here, we report a novel E1A-mutant adenovirus (M6) with antisense HPV16 E6 E7 DNA inserted into the deleted 6.7K/gp19K region of E3. The target effects of M6 on HPV16-positive cervical cancer cells were evaluated in vivo and in vitro. By using cytopathic effect (CPE) and viral replication assays, we verified M6 was competent to selectively replicate in cervical cancer cells in vitro. Moreover, we found infection of M6 was able to inhibit the expression of HPV16 E6 and E7 oncogenes and induce apoptosis of HPV16-positive cervical cancer cells. Further analysis in vitro revealed that the invasive ability of SiHa cells was significantly inhibited by M6. To determine if M6 synergized with radiotherapy-induced anti-tumor activity against HPV16-related cancer cells, we transfected SiHa cells with M6 followed by a single exposure to radiation. A significantly suppression of cell growth and induced apoptosis was observed in SiHa cells received M6 transfection combined with radiotherapy. Animal experiments showed that M6 transfection notably improved the survival of tumor-bearing mice in combination with radiotherapy, much superior to that of those treated by Adv5/dE1A plus radiation or M6 alone. These findings indicated the anti-tumoral efficacy of M6 on HPV16-positive cervical cancer cells and its synergic therapeutic application in radiation for cervical cancer. [Copyright &y& Elsevier]

Integration of high-risk human papillomavirus (HR-HPVs) into the host DNA has been proposed as a risk for cervical carcinogenesis. HPV-16 is the predominant high-risk type and its integration ration varied largely in different cervical cancer (CC) samples. The aim of this study was to evaluate the correlation between physical state of HPV16 infection and extent of cervical lesion, as well as the clinical significance of virus existing state. A total of 252 cases of paraffin-embedded blocks derived from cancer precursor lesion and cervical carcinoma samples were detected by HC-II for HR-HPV infection. HPV16 infection was confirmed by PCR and immunohistochemistry for HPV16 E7 simultaneously. The physical state of HPV16 infection were assessed by PCR for 3 overlapping fragments in E2 gene and multiple PCR for E2 gene and E7 gene. The infection ratio of HR-HPV in normal cervical tissue, cervical intraepithelial neoplasia (CIN) I, CIN II, CIN III and cervical cancer were 15.0, 32.8, 54.3, 69.7, 93.8%, respectively. HR-HPV positive samples of 62.8% were infected with HPV16. The integration ratio of HPV16 in CIN III and cervical carcinoma were 35.7 and 58.1% respectively, both of which were significantly higher than that of CIN I and normal cervical tissues. The discrepancy was statistically significant ( P < 0.05). Furthermore, it was observed that persistent virus infection and progression of cervical lesion were more common in CIN I with integrated HPV16 than that with episomal HPV16. The integration ratio of HPV16 was accompanied by an increase in the grade of cervical lesion. The integrated state of HPV16 infection was strongly associated with persistent HPV infection and progression of cervical lesions. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Human papillomavirus type 16 (HPV-16) is a high-risk DNA tumor virus involved in the development of cervical carcinomas. Substantial studies have demonstrated that E6 and E7 oncoproteins of HPV-16 could induce cell proliferation and immortalization. Repression of E6 and/or E7 oncogenes may induce cervical cancer cells to undergo apoptosis or senescence. The purpose of this study was to determine whether activation of the p53 and retinoblastoma (Rb) pathway by HPV-16 E6 and E7 repression was responsible for apoptosis and senescence of cervical cancer cells and to explore the potential of an antisense RNA (AS) transcript for gene therapy of cervical cancer. Method: The antisense RNA directed against HPV-16 E6 and E7 (16AS) was constructed, and its effects on cell apoptosis and senescence of SiHa cervical carcinoma cells harboring HPV-16 were analyzed. The efficiency of 16AS was evaluated with RT-PCR, Western blotting, flow cytometry analysis, Hoechst 33258 staining, senescent cell morphology observation and senescence-associated β-galactosidase staining. Results: The sufficient repression of HPV-16 E6 and E7 oncogenes were achieved in 16AS-transfected SiHa cells, which led to obvious apoptosis and replicative senescence of tumor cells. Furthermore, the downregulation of HPV-16 E6 and E7 by 16AS transfection resulted in remarkable increase of both p53 expression and hypophosphorylated p105Rb level in SiHa cells. Conclusion: These results demonstrate that reduction of E6 and E7 expression is sufficient to induce SiHa cells to undergo apoptosis and senescence and suggest that transfection of cervical cancer cells with HPV-16 E6 and E7 antisense RNA is a potential approach to treat HPV-16-positive cervical cancers. [Copyright &y& Elsevier]

Abstract: Objective : Human papillomavirus (HPV) is the essential causative factor in cervical carcinogenesis, and apoptosis inhibition is one of the key features of HPV-induced malignant transformation. This study is to investigate the possible cause–effect association between high-risk HPV and cellular FLICE-like inhibitory protein (c-FLIP), an important apoptosis regulator, during cervical carcinogenesis. Methods : A series of 80 archival samples, including 20 squamous cervical carcinomas (SCC) 54 cervical intraepithelial neoplasia (CIN) lesions and 6 normal cervical tissues, were subjected for c-FLIP immunohistochemical staining and HPV HC-II analysis. Typing HPV-16 infection was analyzed by the polymerase chain reaction (PCR), and its status was assessed with the integrity and disruption of the HPV-16 E2 gene, which was amplified in three overlapping fragments. Results : The types of HR-HPV infection and E2 disruption were associated closely with cervical lesion severity. There was a significant relationship between lesion grade and c-FLIP expression level. c-FLIP overexpression was also closely associated with HR-HPV infection and its integration status. Multivariate regression analysis revealed c-FLIP as a strong independent predictor for CIN, with 100% PPV, and showed 90.9% PPV in detecting HR-HPV, and remained a significance factor to rule out which case has no HR-HPV integration, with a 94.7% sensitivity and a 90.0% NPV. Conclusions : The present data approved that c-FLIP overexpression is related significantly to the presence of HR-HPV infection and its integration status during progression of cervical squamous cell cancer and confirmed the role of c-FLIP as an early marker of cervical carcinogenesis. [Copyright &y& Elsevier]

It has been demonstrated that miR-145 is expressed in primordial follicles and modulates the initiation of primordial follicle development. We aimed to explore the function of miR-145 in mouse granulosa cells (mGCs). The proliferation and differentiation of GCs were examined via MTT, EDU assay, QRT-PCR, ELISA and electron microscope analysis. The target of miR-145 was determined by bioinformatics analysis and luciferase reporter assay and the molecular mechanisms were examined via western blot and quantitative Real-Time RT-PCR. We proved that down-regulation of miR-145 could inhibit GCs proliferation and differentiation. In addition, we provided evidence that Crkl was the target gene of miR-145. The miR-145 antagomir caused an increase in Crkl expression and activation of the JNK/p38 MAPK pathway. Overexpression of Crkl with pEGFP-N1- Crkl vector inhibited GCs differentiation and progesterone synthesis as well as activation of the JNK/p38 MAPK pathway. Our study shows that miR-145 targets Crkl and through the JNK/p38 MAPK signaling pathway promotes the GCs proliferation, differentiation, and steroidogenesis. MiR-145 may play an important role in the ovarian physiology and pathology. [ABSTRACT FROM AUTHOR]

The authors would like to correct figure, as the error was introduced in the preparation of this figure for publication. We sincerely apologize for having this error in the article, the authors have provided corrected version of figure here. [ABSTRACT FROM AUTHOR]