Icaritin, a compound from Epimedium Genus, has selective estrogen receptor (ER) modulating activities, and posses anti-tumor activity. Here, we examined icaritin effect on cell growth of human endometrial cancer Hec1A cells and found that icaritin potently inhibited proliferation of Hec1A cells. Icaritin-inhibited cell growth was associated with increased levels of p21 and p27 expression and reduced cyclinD1 and cdk 4 expression. Icaritin also induced cell apoptosis accompanied by activation of caspases as evidenced by the cleavage of endogenous substrate Poly (ADP-ribose) polymerase (PARP) and cytochrome c release, which was abrogated by pretreatment with the pan-caspase inhibitor z-VAD-fmk. Icaritin treatment also induced expression of pro-apoptotic protein Bax with a concomitant decrease of Bcl-2 expression. Furthermore, icaritin induced sustained phosphorylation of extracellular signal-regulated kinase1/2 (the MAPK/ ERK1/2) in Hec1A cells and U0126, a specific MAP kinase kinase (MEK1/2) inhibitor, blocked the ERK1/2 activation by icaritin and abolished the icaritin-induced growth inhibition and apoptosis. Our results demonstrated that icaritin induced sustained ERK 1/2 activation and inhibited growth of endometrial cancer Hec1A cells, and provided a rational for preclinical and clinical evaluation of icaritin for endometrial cancer therapy.

Microtubules are regulated by posttranslational modifications (PTMs) of tubulin. The ligation and cleavage of the C-terminal tyrosine of α tubulin impact microtubule functions during mitosis, cardiomyocyte contraction, and neuronal processes. Tubulin tyrosination and detyrosination are mediated by tubulin tyrosine ligase (TTL) and the recently discovered tubulin detyrosinases, vasohibin 1 and 2 (VASH1 and VASH2) bound to the small vasohibin-binding protein (SVBP). Here, we report the crystal structures of human VASH1–SVBP alone, in complex with a tyrosine-derived covalent inhibitor, and bound to the natural product parthenolide. The structures and subsequent mutagenesis analyses explain the requirement for SVBP during tubulin detyrosination, and reveal the basis for the recognition of the C-terminal tyrosine and the acidic α tubulin tail by VASH1. The VASH1–SVBP–parthenolide structure provides a framework for designing more effective chemical inhibitors of vasohibins, which can be valuable for dissecting their biological functions and may have therapeutic potential.

During the oogenesis of Xenopus laevis, oocytes accumulate maternal materials for early embryo development. As the transcription activity of the oocyte is silenced at the fully grown stage and the global genome is reactivated only by the mid-blastula embryo stage, the translation of maternal mRNAs accumulated during oocyte growth should be accurately regulated. Previous evidence has illustrated that the poly(A) tail length and RNA binding elements mediate RNA translation regulation in the oocyte. Recently, RNA methylation has been found to exist in various systems. In this study, we sequenced the N 6 -methyladenosine (m 6 A) modified mRNAs in fully grown germinal vesiclestage and metaphase II-stage oocytes. As a result, we identified 4207 mRNAs with m 6 A peaks in germinal vesicle-stage or metaphase II-stage oocytes. When we integrated the mRNA methylation data with transcriptome and proteome data, we found that the highly methylated mRNAs showed significantly lower protein levels than those of the hypomethylated mRNAs, although the RNA levels showed no significant difference. We also found that the hypomethylated mRNAs were mainly enriched in the cell cycle and translation pathways, whereas the highly methylated mRNAs were mainly associated with protein phosphorylation. Our results suggest that oocyte mRNA methylation can regulate cellular translation and cell division during oocyte meiotic maturation and early embryo development.During oogenesis in animals like Xenopus laevis, mouse, and human, germinal vesicle (GV)-stage 3 oocytes gradually achieve maximum size, and genomic transcription activity is silenced (1, 2). After that, the fully grown GV oocytes resume meiosis and develop to metaphase of the second meiosis (MII) stage. Then the oocyte is fertilized by sperm to form a zygote. The zygote starts mitosis, and embryo development is initiated. As embryo genomic transcription is not reactivated until embryo development to the mid-blastula stage for X. laevis (3) or the 2-cell to 16-cell stages for mammals (4 -7), oocytes or embryos need the maternal RNAs accumulated during oocyte growth to support new protein synthesis.As the synthesis of new proteins such as cyclins is of importance for the meiotic and mitotic events, the translation of maternal mRNAs during oocyte meiotic maturation and early embryo development should be precisely controlled. Previous data have shown that there are mainly two methods for cells to control the maternal mRNA translation in oocyte or early embryo protein binding to the cytoplasmic polyadenylation elements (CPEs) in maternal mRNAs and controlling the polyadenylation of the mRNA poly(A) tails (8). In most cases, shortened poly(A) tails of mRNAs repress their translation, whereas elongated poly(A) tails activate translation (9). The poly(A) tail length of oocyte mRNA is mainly associated with cytoplasmic polyadenylation, which is controlled by RNA binding proteins and associated proteins. The maternal mRNAs, whose 3Ј UTR contains a cis-element CPE, could be bonded by CPE binding...

Cyclin B3 is a relatively new member of the cyclin family whose functions are little known. We found that depletion of cyclin B3 inhibited metaphase-anaphase transition as indicated by a well-sustained MI spindle and cyclin B1 expression in meiotic oocytes after extended culture. This effect was independent of spindle assembly checkpoint activity, since both Bub3 and BubR1 signals were not observed at kinetochores in MI-arrested cells. The metaphase I arrest was not rescued by either Mad2 knockdown or cdc20 overexpression, but it was rescued by securin RNAi. We conclude that cyclin B3 controls the metaphase-anaphase transition by activating APC/C cdc20 in meiotic oocytes, a process that does not rely on SAC activity.

BackgroundAneuploidy is one of the major factors that result in low efficiency in human infertility treatment by in vitro fertilization (IVF). The development of DNA microarray technology allows for aneuploidy screening by analyzing all 23 pairs of chromosomes in human embryos. All chromosome screening for aneuploidy is more accurate than partial chromosome screening, as errors can occur in any chromosome. Currently, chromosome screening for aneuploidy is performed in developing embryos, mainly blastocysts. It has not been performed in arrested embryos and/or compared between developing embryos and arrested embryos from the same IVF cycle.MethodsThe present study was designed to examine all chromosomes in blastocysts and arrested embryos from the same cycle in patients of advanced maternal ages. Embryos were produced by routine IVF procedures. A total of 90 embryos (45 blastocysts and 45 arrested embryos) from 17 patients were biopsied and analyzed by the Agilent DNA array platform.ResultsIt was found that 50% of the embryos developed to blastocyst stage; however, only 15.6% of the embryos (both blastocyst and arrested) were euploid, and most (84.4%) of the embryos had chromosomal abnormalities. Further analysis indicated that 28.9% of blastocysts were euploid and 71.1% were aneuploid. By contrast, only one (2.2%) arrested embryo was euploid while others (97.8%) were aneuploid. The prevalence of multiple chromosomal abnormalities in the aneuploid embryos was also higher in the arrested embryos than in the blastocysts.ConclusionsThese results indicate that high proportions of human embryos from patients of advanced maternal age are aneuploid, and the arrested embryos are more likely to have abnormal chromosomes than developing embryos.

SummaryChromosome segregation in mammalian oocyte meiosis is an error-prone process, and any mistake in this process may result in aneuploidy, which is the main cause of infertility, abortion and many genetic diseases. It is now well known that shugoshin and protein phosphatase 2A (PP2A) play important roles in the protection of centromeric cohesion during the first meiosis. PP2A can antagonize the phosphorylation of rec8, a member of the cohesin complex, at the centromeres and thus prevent cleavage of rec8 and so maintain the cohesion of chromatids. SETb is a protein that physically interacts with shugoshin and inhibits PP2A activity. We thus hypothesized that SETb might regulate cohesion protection and chromosome segregation during oocyte meiotic maturation. Here we report for the first time the expression, subcellular localization and functions of SETb during mouse oocyte meiosis. Immunoblotting analysis showed that the expression level of SETb was stable from the germinal vesicle stage to the MII stage of oocyte meiosis. Immunofluorescence analysis showed SETb accumulation in the nucleus at the germinal vesicle stage, whereas it was targeted mainly to the inner centromere area and faintly localized to the interchromatid axes from germinal vesicle breakdown to MI stages. At the MII stage, SETb still localized to the inner centromere area, but could relocalize to kinetochores in a process perhaps dependent on the tension on the centromeres. SETb partly colocalized with PP2A at the inner centromere area. Overexpression of SETb in mouse oocytes caused precocious separation of sister chromatids, but depletion of SETb by RNAi showed little effects on the meiotic maturation process. Taken together, our results suggest that SETb, even though it localizes to centromeres, might not be essential for chromosome separation during mouse oocyte meiotic maturation, although its forced overexpression causes premature chromatid separation.

BackgroundThe adverse effects of maternal diabetes on oocyte maturation and embryo development have been reported.MethodsIn this study, we used time-lapse live cell imaging confocal microscopy to investigate the dynamic changes of ER and the effects of diabetes on the ER’s structural dynamics during oocyte maturation, fertilization and early embryo development.ResultsWe report that the ER first became remodeled into a dense ring around the developing MI spindle, and then surrounded the spindle during migration to the cortex. ER reorganization during mouse early embryo development was characterized by striking localization around the pronuclei in the equatorial section, in addition to larger areas of fluorescence deeper within the cytoplasm. In contrast, in diabetic mice, the ER displayed a significantly higher percentage of homogeneous distribution patterns throughout the entire ooplasm during oocyte maturation and early embryo development. In addition, a higher frequency of large ER aggregations was detected in GV oocytes and two cell embryos from diabetic mice.ConclusionsThese results suggest that the diabetic condition adversely affects the ER distribution pattern during mouse oocyte maturation and early embryo development.

Ppp2r1a encodes the scaffold subunit Aalpha of protein phosphatase 2A (PP2A), which is an important and ubiquitously expressed serine threonine phosphatase family and plays a critical role in many fundamental cellular processes. To identify the physiological role of PP2A in female germ cell meiosis, we selectively disrupted Ppp2r1a expression in oocytes by using the Cre-Loxp conditional knockout system. Here we report for the first time that oocyte-specific deletion of Ppp2r1a led to severe female subfertility without affecting follicle survival, growth, and ovulation. PP2A-Aalpha was essential for regulating oocyte meiotic maturation because depletion of PP2A-Aalpha facilitated germinal vesicle breakdown, causing elongation of the MII spindle and precocious separation of sister chromatids. The resulting eggs had high risk of aneuploidy, though they could be fertilized, leading to defective embryonic development and thus subfertility. Our findings provide strong evidence that PP2A-Aalpha within the oocyte plays an indispensable role in oocyte meiotic maturation, though it is dispensable for folliculogenesis in the mouse ovary.