Melatonin prevents oocyte deterioration due to cotinine exposure in mice†.

Levels of cotinine, a major metabolite of nicotine, have been positively correlated with risks of cigarette smoking-related diseases. Melatonin is synthesized by the pineal gland and has been demonstrated to be beneficial to oocyte maturation due to its antioxidative activity. In this study, we investigated the effects of cotinine on mouse oocyte meiosis and the protective roles of melatonin in vitro and in vivo. The results showed that cotinine exposure caused defects in the first polar body extrusion and reduced parthenogenetic activation in in vitro-matured oocytes. Additionally, cotinine exposure increased the level of oxidative stress, which resulted in aberrant actin distribution, abnormal spindle morphology, chromosome misalignment, and even oocyte aneuploidy. Simultaneously, cotinine exposure decreased the mitochondrial membrane potential and antioxidant gene expression and increased apoptosis-related gene expression. However, all these toxic effects of cotinine could be reversed after the addition of melatonin, and the mechanism may be a decrease in reactive oxygen species production. In conclusion, cotinine causes poor oocyte quality, which could be rescued by melatonin supplementation during meiotic maturation in mouse oocytes.

Compound Kushen injection inhibits EMT of gastric cancer cells via the PI3K/AKT pathway.

BACKGROUND: The effective components contained in compound Kushen injection (CKI) and the genes and signalling pathways related to gastric cancer (GC) were analyzed through the network pharmacology method of traditional Chinese medicine, and various possible mechanisms by which CKI affects the proliferation, differentiation, survival, and metastasis of GC cells were discussed. The PI3K/AKT signalling pathway is considered to be one of the most important pathways targeted by CKI in the regulation of GC cells. The implementation of related cell experiments also confirmed the information we revealed. METHODS: Effective drug components of Kushen and Baituling in CKI were identified from the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP). Genes related to GC were identified using the GENECARD and OMIM databases. The common target genes related to the effective components of the drug and GC were identified using the intersection method and visualized using software. A protein-protein interaction network (PPI) was established using STRING online software to confirm the key genes. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to predict the key pathways of CKI in GC treatment. BGC-803 and MKN-28 GC cells were used to verify the signalling pathway. Cell proliferation, apoptosis, migration ability, and invasion ability were assessed using CCK8, flow cytometry, scratch, and transwell assays. Immunofluorescence assays and western blotting were used to detect the expression of related proteins. RESULTS: CKI regulated GC cells through 35 effective drug components of GC-related target genes. In total, 194 genes were common targets of CKI and GC. The most significant function of the enriched genes was DNA-binding transcription activator activity as demonstrated by GO enrichment analysis. The metabolic pathway with the highest enrichment was the PI3K/AKT signalling pathway as demonstrated by KEGG enrichment analysis. Our cell experimental evidence also shows that CKI inhibits GC cell growth and migration and induce GC cell apoptosis. In addition, CKI inhibits the EMT process in GC cells through the PI3K/AKT signalling pathway. CONCLUSION: AKT1 is a key gene for CKI treatment of GC. CKI inhibited GC cell growth and migration and induced GC cell apoptosis. In addition, CKI regulated the EMT process in GC cells through the PI3K/AKT signalling pathway.

The cohesion establishment factor Esco1 acetylates α-tubulin to ensure proper spindle assembly in oocyte meiosis.

Esco1 has been reported to function as a cohesion establishment factor that mediates chromosome cohesion and segregation in mitotic cells. However, its exact roles in meiosis have not been clearly defined. Here, we document that Esco1 is expressed and localized to both the nucleus and cytoplasm during mouse oocyte meiotic maturation. Depletion of Esco1 by siRNA microinjection causes the meiotic progression arrest with a severe spindle abnormality and chromosome misalignment, which is coupled with a higher incidence of the erroneous kinetochore-microtubule attachments and activation of spindle assembly checkpoint. In addition, depletion of Esco1 leads to the impaired microtubule stability shown by the weakened resistance ability to the microtubule depolymerizing drug nocodazole and the decreased level of acetylated α-tubulin. Conversely, overexpression of Esco1 causes hyperacetylation of α-tubulin and spindle defects. Moreover, we find that Esco1 binds to α-tubulin and is required for its acetylation. The reduced acetylation level of α-tubulin in Esco1-depleted oocytes can be restored by the ectopic expression of exogenous wild-type Esco1 but not enzymatically dead Esco1-G768D. Purified wild-type Esco1 instead of mutant Esco1-G768D acetylates the synthesized peptide of α-tubulin in vitro. Collectively, our data assign a novel function to Esco1 as a microtubule regulator during oocyte meiotic maturation beyond its conventional role in chromosome cohesion.

Postovulatory aging causes the deterioration of porcine oocytes via induction of oxidative stress.

Negative effects of postovulatory aging on fertilization ability and subsequent embryo development have been reported in rodents; however, the molecular and cellular changes during this process have not been fully defined. Here, we used porcine oocytes, a model that is physiologically and developmentally similar to humans, to explore the molecular mechanisms that underlie how postovulatory aging affects oocyte quality and fertilization capacity. We found that postovulatory aging caused the morphologic change of porcine oocytes by exhibiting the incompact expansion of cumulus cells and an increased occurrence of fragmentation. Aging also impaired oocyte quality by disrupting organelle structures, including the spindle assembly, actin polymerization, and mitochondrial integrity. Moreover, postovulatory aging led to the abnormal distribution of cortical granules and ovastacin, which, in turn, resulted in defective sperm binding and consequently compromised fertilization potential. Of note, we observed that postovulatory aging induced oxidative stress with a high level of reactive oxygen species and apoptotic rate in oocytes, thereby resulting in the deterioration of critical factors in the maintenance of oocyte quality and fertilization capacity. Taken together, our findings demonstrate that postovulatory aging perturbs a variety of molecular and cellular changes in porcine oocytes by inducing oxidative stress.-Miao, Y., Zhou, C., Cui, Z., Zhang, M., ShiYang, X., Lu, Y., Xiong, B. Postovulatory aging causes the deterioration of porcine oocytes via induction of oxidative stress.

BaP exposure causes oocyte meiotic arrest and fertilization failure to weaken female fertility.

Benzo[a]pyrene (BaP) is a ubiquitous environmental pollutant and carcinogen that is frequently found in particulate matter, with a diameter of ≤2.5 µm (PM2.5). It has been reported to interrupt the normal reproductive system, but the exact molecular basis has not been clearly defined. To understand the underlying mechanisms regarding how BaP exposure disrupts female fertility, we evaluated oocyte quality by assessing the critical regulators and events during oocyte meiotic maturation and fertilization. We found that BaP exposure compromised the mouse oocyte meiotic progression by disrupting normal spindle assembly, chromosome alignment, and kinetochore-microtubule attachment, consequently leading to the generation of aneuploid eggs. In addition, BaP administration significantly decreased the fertilization rate of mouse eggs by reducing the number of sperm binding to the zona pellucida, which was consistent with the premature cleavage of N terminus of zona pellucida sperm-binding protein 2 and precocious exocytosis of ovastacin. Furthermore, BaP exposure interfered with the gamete fusion process by perturbing the localization and protein level of Juno. Notably, we found that BaP exposure induced oxidative stress with an increased level of reactive oxygen species and apoptosis in oocytes and thereby led to the deterioration of critical regulators and events during oocyte meiotic progression and fertilization. Our data document that BaP exposure reduces female fertility via impairing oocyte maturation and fertilization ability induced by oxidative stress and early apoptosis in murine models.-Zhang, M., Miao, Y., Chen, Q., Cai, M., Dong, W., Dai, X., Lu, Y., Zhou, C., Cui, Z., Xiong, B. BaP exposure causes oocyte meiotic arrest and fertilization failure to weaken female fertility.

Cohesin acetyltransferase Esco2 regulates SAC and kinetochore functions via maintaining H4K16 acetylation during mouse oocyte meiosis.

Sister chromatid cohesion, mediated by cohesin complex and established by the acetyltransferases Esco1 and Esco2, is essential for faithful chromosome segregation. Mutations in Esco2 cause Roberts syndrome, a developmental disease characterized by severe prenatal retardation as well as limb and facial abnormalities. However, its exact roles during oocyte meiosis have not clearly defined. Here, we report that Esco2 localizes to the chromosomes during oocyte meiotic maturation. Depletion of Esco2 by morpholino microinjection leads to the precocious polar body extrusion, the escape of metaphase I arrest induced by nocodazole treatment and the loss of BubR1 from kinetochores, indicative of inactivated SAC. Furthermore, depletion of Esco2 causes a severely impaired spindle assembly and chromosome alignment, accompanied by the remarkably elevated incidence of defective kinetochore-microtubule attachments which consequently lead to the generation of aneuploid eggs. Notably, we find that the involvement of Esco2 in SAC and kinetochore functions is mediated by its binding to histone H4 and acetylation of H4K16 both in vivo and in vitro. Thus, our data assign a novel meiotic function to Esco2 beyond its role in the cohesion establishment during mouse oocyte meiosis.

The protective role of melatonin in porcine oocyte meiotic failure caused by the exposure to benzo(a)pyrene.

STUDY QUESTION: Does melatonin restore the benzo(a)pyrene (BaP)-induced meiotic failure in porcine oocytes? SUMMARY ANSWER: Melatonin effectively inhibits the increased reactive oxygen species (ROS) level and apoptotic rate in BaP-exposed porcine oocytes to recover the meiotic failure. WHAT IS KNOWN ALREADY: BaP, a widespread environmental carcinogen found in particulate matter, 2.5 µm or less (PM2.5), has been shown to have toxicity at the level of the reproductive systems. BaP exposure disrupts the steroid balance, alters the expression of ovarian estrogen receptor and causes premature ovarian failure through the rapid depletion of the primordial follicle pool. In addition, acute exposure to BaP has transient adverse effects on the follicle growth, ovulation and formation of corpora lutea, which results in transient infertility. STUDY DESIGN, SIZE, DURATION: Porcine oocytes were randomly assigned to control, BaP-exposed and melatonin-supplemented groups. BaP was dissolved in dimethylsulphoxide and diluted to a final concentration of 50, 100 or 250 µM with maturation medium, respectively. Melatonin was dissolved in the absolute ethanol and diluted with maturation medium to a final concentration of 1 nM, 100 nM, 10 µM and 1 mM, respectively. The in vitro cultured oocytes from each group after treatment were applied to the subsequent analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Acquisition of oocyte meiotic competence was assessed using immunostaining, fluorescent intensity quantification and/or immunoblotting to analyse the cytoskeleton assembly, mitochondrial integrity, cortical granule dynamics, ovastacin distribution, ROS level and apoptotic rate. Fertilization ability of oocytes was examined by sperm binding assay and IVF. MAIN RESULTS AND THE ROLE OF CHANCE: BaP exposure resulted in the oocyte meiotic failure (P = 0.001) via impairing the meiotic apparatus, showing a prominently defective spindle assembly (P = 0.003), actin dynamics (P < 0.001) and mitochondrion integrity (P < 0.001). In addition, BaP exposure caused the abnormal distribution of cortical granules (P < 0.001) and ovastacin (P = 0.003), which were consistent with the observation that fewer sperm bound to the zona pellucida surrounding the unfertilized BaP-exposed eggs (P < 0.001), contributing to the fertilization failure (P < 0.001). Conversely, melatonin supplementation recovered, at least partially, all the meiotic defects caused by BaP exposure through inhibiting the rise in ROS level (P = 0.015) and apoptotic rate (P = 0.001). LIMITATIONS, REASONS FOR CAUTION: We investigated the negative impact of BaP on the oocyte meiotic maturation in vitro, but not in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Our findings not only deeply clarify the potential mechanisms of BaP-induced oocyte meiotic failure, but also extend the understanding about how environmental pollutants influence the reproductive systems in humans. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the National Natural Science Foundation of China (31571545) and the Natural Science Foundation of Jiangsu Province (BK20150677). The authors have no conflict of interest to disclose.

Cullin9 protects mouse eggs from aneuploidy by controlling microtubule dynamics via Survivin.

The Cullin9 gene encodes a putative E3 ligase that serves a wide variety of biological functions in mitosis, whereas its roles in meiosis have not yet clearly defined. Here, we report that Cullin9 accumulates on the spindle apparatus and colocalizes with the microtubule fibers during mouse oocyte meiotic maturation. Depletion of Cullin9 by morpholino microinjection results in a remarkably higher rate of disorganized spindles and misaligned chromosomes in oocytes, which is coupled with the impaired kinetochore-microtubule attachments. Resultantly, the incidence of aneuploid eggs significantly increases in Cullin9-depleted oocytes. Moreover, we show that Cullin9 controls Survivin's protein level during meiotic maturation, and thus regulates microtubule stability in oocytes. Thus, our study assigns a new meiotic function to Cullin9 and reveals that it prevents mouse eggs from aneuploidy by regulating microtubule dynamics via Survivin.

Melatonin improves the fertilization ability of post-ovulatory aged mouse oocytes by stabilizing ovastacin and Juno to promote sperm binding and fusion.

STUDY QUESTION: What are the underlying mechanisms of the decline in the fertilization ability of post-ovulatory aged oocytes? SUMMARY ANSWER: Melatonin improves the fertilization ability of post-ovulatory aged oocytes by reducing aging-induced reactive oxygen species (ROS) levels and inhibiting apoptosis and by maintaining the levels and localization of the fertilization proteins, ovastacin and Juno. WHAT IS KNOWN ALREADY: Following ovulation, the quality of mammalian metaphase II oocytes irreversibly deteriorates over time with a concomitant loss of fertilization ability. Melatonin has been found to prevent post-ovulatory oocyte aging and extend the window for optimal fertilization in mice. STUDY DESIGN, SIZE, DURATION: Mouse oocytes were randomly assigned to three groups and aged in vitro for 0, 6, 12 and 24 h, respectively. Increasing concentrations of melatonin (10-9 M, 10-7 M, 10-5 M and 10-3 M) were added to the 24 h aging group. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sperm binding assays, in-vitro fertilization, immunofluorescent staining and western blotting were performed to investigate key regulators and events during fertilization of post-ovulatory aged mouse oocytes. MAIN RESULTS AND THE ROLE OF CHANCE: We found that the actin cap which promotes a cortical granule (CG) free domain is disrupted with a re-distribution of CGs in the subcortex of aged oocytes. Ovastacin, a CG metalloendoprotease, is mis-located and prematurely exocytosed in aged oocytes with subsequent cleavage of the zona pellucida protein ZP2. This disrupts the sperm recognition domain and dramatically reduces the number of sperm binding to the zona pellucida. The abundance of Juno, the sperm receptor on the oocyte membrane, also is reduced in aged oocytes. Exposure of aged oocytes to melatonin significantly elevates in-vitro fertilization rates potentially by rescuing the above age-associated defects of fertilization, and reducing ROS and inhibiting apoptosis. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: We explored the mechanisms of the decline in fertilization ability decline in aged mouse oocytes, in vitro but not in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Our findings may contribute to the development a more efficient method, involving melatonin, for improving IVF success rates. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Natural Science Foundation (31571545) and the Natural Science Foundation of Jiangsu Province (BK20150677). The authors have no conflict of interest to disclose.

Melatonin protects oocyte quality from Bisphenol A-induced deterioration in the mouse.

Bisphenol A (BPA) has been reported to adversely affect the mammalian reproductive system in both sexes. However, the underlying mechanisms regarding how BPA disrupts the mammalian oocyte quality and how to prevent it have not been fully defined. Here, we document that BPA weakens oocyte quality by impairing both oocyte meiotic maturation and fertilization ability. We find that oral administration of BPA (100 µg/kg body weight per day for 7 days) compromises the first polar body extrusion (78.0% vs 57.0%, P<.05) by disrupting normal spindle assembly, chromosome alignment, and kinetochore-microtubule attachment. This defect could be remarkably ameliorated (76.7%, P<.05) by concurrent oral administration of melatonin (30 mg/kg body weight per day for 7 days). In addition, BPA administration significantly decreases the fertilization rate of oocytes (87.2% vs 41.1%, P<.05) by reducing the number of sperm binding to the zona pellucida, which is consistent with the premature cleavage of ZP2 as well as the mis-localization and decreased protein level of ovastacin. Also, the localization and protein level of Juno, the sperm receptor on the egg membrane, are strikingly impaired in BPA-administered oocytes. Finally, we show that melatonin administration substantially elevates the in vitro fertilization rate (63.0%, P<.05) by restoring above defects of fertilization proteins and events, which might be mediated by the improvement of oocyte quality via reduction of ROS levels and inhibition of apoptosis. Collectively, our data reveal that melatonin has a protective action against BPA-induced deterioration of oocyte quality in mice.

Extracellular signal-regulated protein kinase 5 modulates the spindle assembly to coordinate the oocyte meiotic maturation.

Extracellular signal-regulated protein kinase 5 (Erk5), a member of the mitogen-activated protein kinase (MAPK) family, is ubiquitously expressed in all eukaryotic cells and is implicated in the various mitotic processes such as cell survival, proliferation, migration, and differentiation. However, the potential functional roles of Erk5 in oocyte meiosis have not been fully determined. In this study, we document that ERK5 participates in the meiotic maturation of mouse oocytes by regulating the spindle assembly to ensure the meiotic progression. We unexpectedly found that phosphorylated ERK5 was localized in the spindle pole region at metaphase I and II stages by immunostaining analysis. Inhibition of ERK5 activity using its specific inhibitor XMD8-92 dramatically reduced the incidence of first polar body extrusion. In addition, inhibition of ERK5 evoked the spindle assembly checkpoint to arrest oocytes at metaphase I stage by impairing the spindle assembly, chromosome alignment and kinetochore-microtubule attachment. Mechanically, over-strengthened microtubule stability was shown to disrupt the microtubule dynamics and thus compromise the spindle assembly in ERK5-inhibited oocytes. Conversely, overexpression of ERK5 caused decreased level of acetylated α-tubulin and spindle defects. Collectively, we conclude that ERK5 plays an important role in the oocyte meiotic maturation by regulating microtubule dynamics and spindle assembly.

Toxic effects of 4-methylimidazole on the maturation and fertilization of mouse oocytes.

Individuals of all ages, including children and teenagers, consume 4-methylimidazole (4-MI) in their food. 4-MI is a caramel-colored waste product that has previously been linked to human carcinogenesis and has shown possible signs of reproductive toxicity. This study aimed to determine whether 4-MI is harmful to oocytes during meiosis and fertilization. Female mice were intragastrically administered 0, 50, or 100 mg/kg body weight of 4-MI daily for 10 days. We found that 4-MI affects the quality of oocytes by affecting their meiotic ability and fertility potential. Specifically, 4-MI rendered the meiotic spindles and chromosomes less stable, which halted oocyte maturation and resulted in aneuploidy. 4-MI also slowed the decrease in the levels of cortical granules and their component ovastacin; consequently, sperms could not be bound and fertilization could not occur. We also found that mitochondrial dysfunction was associated with oocytes deterioration. This led to reactive oxygen species accumulation and cell death. Altogether, our findings reveal that the poor condition of oocytes subjected to 4-MI is primarily attributable to mitochondrial malfunction and redox alterations.

Dibutyltin dichloride exposure affects mouse oocyte quality by inducing spindle defects and mitochondria dysfunction.

Dibutyltin dichloride (DBTCl) is a widespread environmental pollutant that is frequently employed as a light and heat sustainer for polyvinyl chloride (PVC) plastics and is a teratogen in vivo. Nevertheless, its destructiveness in mammalian oocytes remains unclear. This study highlighted the consequences of DBTCl vulnerability on mouse oocyte. Our results revealed that exposure to 5.0 mg/kg/day of DBTCl for ten days reduced the number of mature follicles and oocytes in the ovaries and inhibited the meiotic maturation of oocytes. Single-cell transcriptomic analysis indicated that DBTCl exposure interfered with the expression of more than 400 genes in oocytes, including those involved in multiple biological pathways. Specifically, DBTCl exposure impaired spindle assembly and chromosome alignment. In addition, DBTCl exposure caused mitochondrial dysfunction, which led to the accumulation of reactive oxygen species (ROS) and induced apoptosis. In summary, our study illustrates that mitochondrial dysfunction and redox perturbation are the major causes of the reduced quality of oocytes exposed to DBTCl.

Mogroside V ameliorates the oxidative stress-induced meiotic defects in porcine oocytes in vitro.

It has been reported that environmental factors, such as industrial pollution, environmental toxins, environmental hormones, and global warming contribute to the oxidative stress-induced deterioration of oocyte quality and female fertility. However, the prevention or improvement approaches have not been fully elucidated. Here, we explored the mechanism regarding how Mogroside V (MV), a main extract of Siraitia grosvenorii, improves the oxidative stress-induced meiotic defects in porcine oocytes. Our results showed that MV supplementation restores the defective oocyte maturation and cumulus cell expansion caused by H2O2 treatment. We further found that MV supplementation promoted the oocyte cytoplasmic maturation through preventing cortical granules from the aberrant distribution, and drove the nuclear maturation by maintaining the cytoskeleton structure. Notably, our single-cell RNA sequencing data indicated that H2O2-treated oocytes led to the oxidative stress primarily through two pathways 'meiosis' and 'oxidative phosphorylation'. Lastly, we evaluated the effects of MV supplementation on the mitochondrial distribution pattern and membrane potential in H2O2-treated oocytes, revealing that MV supplementation eliminated the excessive ROS induced by the mitochondrial abnormalities and consequently suppressed the apoptosis. In conclusion, our study demonstrates that MV supplementation is an effective approach to ameliorate the oxidative stress-induced meiotic defects via recovering the mitochondrial integrity in porcine oocytes.

Transglutaminase 2 crosslinks zona pellucida glycoprotein 3 to prevent polyspermy.

Soon after fertilization, the block mechanisms are developed in the zona pellucida (ZP) and plasma membrane of the egg to prevent any additional sperm from binding, penetration, and fusion. However, the molecular basis and underlying mechanism for the post-fertilization block to sperm penetration through ZP has not yet been determined. Here, we find that transglutaminase 2 (Tgm2), an enzyme that catalyzes proteins by the formation of an isopeptide bond within or between polypeptide chains, crosslinks zona pellucida glycoprotein 3 (ZP3) to result in the ZP hardening after fertilization and thus prevents polyspermy. Tgm2 abundantly accumulates in the subcortical region of the oocytes and vanishes upon fertilization. Both inhibition of Tgm2 activity in oocytes by the specific inhibitor in vitro and genetic ablation of Tgm2 in vivo cause the presence of additional sperm in the perivitelline space of fertilized eggs, consequently leading to the polyploid embryos. Biochemically, recombinant Tgm2 binds to and crosslinks ZP3 proteins in vitro, and incubation of oocytes with recombinant Tgm2 protein inhibits the polyspermy. Altogether, our data identify Tgm2 as a participant of zona block to the post-fertilization sperm penetration via hardening ZP surrounding fertilized eggs, extending our current understanding about the molecular basis of block to polyspermy.

Cyst stem cell lineage eIF5 non-autonomously prevents testicular germ cell tumor formation via eIF1A/eIF2γ-mediated pre-initiation complex.

BACKGROUND: Stem cell niche maintains stem cell population identity and is essential for the homeostasis of self-renewal and differentiation in Drosophila testes. However, the mechanisms of CySC lineage signals-mediated soma-germline communications in response to external stimuli are unclear. METHODS: Pre-initiation complex functions were evaluated by UAS-Gal4-mediated cell effects. RNA sequencing was conducted in NC and eIF5 siRNA-treated cells. Genetic interaction analysis was used to indicate the relationships between eIF5 and eIF1A/eIF2γ in Drosophila testes. RESULTS: Here, we demonstrated that in CySCs, translation initiation factor eIF5 mediates cyst cell differentiation and the non-autonomously affected germ cell differentiation process. CySCs lacking eIF5 displayed unbalanced cell proliferation and apoptosis, forming testicular germ cell tumors (TGCTs) during spermatogenesis. eIF5 transcriptional regulation network analysis identified multiple metabolic processes and several key factors that might be involved in germ cell differentiation and TGCT formation. Importantly, knockdown of eIF1A and eIF2γ, key components of pre-initiation complex, mimicked the phenotype of knocking down eIF5 in the stem cell niche of Drosophila testes. Genetic interaction analysis indicated that eIF5 was sufficient to rescue the phenotype of tumorlike structures induced by down-regulating eIF1A or eIF2γ in CySCs. CONCLUSIONS: These findings demonstrated that CySC lineage eIF5, together with eIF1A or eIF2γ, mediates soma-germline communications for the stem cell niche homeostasis in Drosophila testes, providing new insights for the prevention of TGCTs.

HDAC8 drives spindle organization during meiotic maturation of porcine oocytes.

OBJECTIVES: Histone deacetylase 8 (HDAC8) is one of the class I HDAC family proteins, which participates in the neuronal disorders, parasitic/viral infections, tumorigenesis and many other biological processes. However, its potential function during female germ cell development has not yet been fully understood. MATERIALS AND METHODS: HDAC8-targeting siRNA was microinjected into GV oocytes to deplete HDAC8. PCI-34051 was used to inhibit the enzyme activity of HDAC8. Immunostaining, immunoblotting and fluorescence intensity quantification were applied to assess the effects of HDAC8 depletion or inhibition on the oocyte meiotic maturation, spindle/chromosome structure, γ-tubulin dynamics and acetylation level of α-tubulin. RESULTS: We observed that HDAC8 was localized in the nucleus at GV stage and then translocated to the spindle apparatus from GVBD to M II stages in porcine oocytes. Depletion of HDAC8 led to the oocyte meiotic failure by showing the reduced polar body extrusion rate. In addition, depletion of HDAC8 resulted in aberrant spindle morphologies and misaligned chromosomes due to the defective recruitment of γ-tubulin to the spindle poles. Notably, these meiotic defects were photocopied by inhibition of HDAC8 activity using its specific inhibitor PCI-34051. However, inhibition of HDAC8 did not affect microtubule stability as assessed by the acetylation level of α-tubulin. CONCLUSIONS: Collectively, our findings demonstrate that HDAC8 acts as a regulator of spindle assembly during porcine oocyte meiotic maturation.

Insufficiency of melatonin in follicular fluid is a reversible cause for advanced maternal age-related aneuploidy in oocytes.

Age-related decline in female fertility is a common feature that occurs in the fourth decade of women as a result of a reduction in both oocyte quality and quantity [1]. However, strategies to prevent the deterioration of maternal aged oocytes and relevant mechanisms are still underexplored. Here, we find that the reduced abundance of melatonin in the follicular fluid highly correlates with the advanced maternal age-related aneuploidy. Of note, we show that exposure of oocytes from aged mice both in vitro and in vivo to exogenous melatonin not only eliminates the accumulated reactive oxygen species-induced DNA damage and apoptosis, but also suppresses the occurrence of aneuploidy caused by spindle/chromosome defect that is frequently observed in aged oocytes. Importantly, we reveal that melatonin supplementation reverses the defective phenotypes in aged oocytes through a Sirt1/Sod2-dependent mechanism. Inhibition of Sirt1 activity abolishes the melatonin-mediated improvement of aged oocyte quality. Together our findings provide evidence that supplementation of melatonin is a feasible way to protect oocytes from advanced maternal age-related meiotic defects and aneuploidy, demonstrating the potential for improving the quality of oocytes from aged women and the efficiency of assisted reproductive technology.

Casein kinase 2 modulates the spindle assembly checkpoint to orchestrate porcine oocyte meiotic progression.

BACKGROUND: CK2 (casein kinase 2) is a serine/threonine-selective protein kinase that has been involved in a variety of cellular processes such as DNA repair, cell cycle control and circadian rhythm regulation. However, its functional roles in oocyte meiosis have not been fully determined. RESULTS: We report that CK2 is essential for porcine oocyte meiotic maturation by regulating spindle assembly checkpoint (SAC). Immunostaining and immunoblotting analysis showed that CK2 was constantly expressed and located on the chromosomes during the entire oocyte meiotic maturation. Inhibition of CK2 activity by its selective inhibitor CX-4945 impaired the first polar body extrusion and arrested oocytes at M I stage, accompanied by the presence of BubR1 at kinetochores, indicative of activated SAC. In addition, we found that spindle/chromosome structure was disrupted in CK2-inhibited oocytes due to the weakened microtubule stability, which is a major cause resulting in the activation of SAC. Last, we found that the level DNA damage as assessed by γH2A.X staining was considerably elevated when CK2 was inhibited, suggesting that DNA damage might be another critical factor leading to the SAC activation and meiotic failure of oocytes. CONCLUSIONS: Our findings demonstrate that CK2 promotes the porcine oocyte maturation by ensuring normal spindle assembly and DNA damage repair.

Excessive splenic volume is an unfavorable prognostic factor in patients with non-small cell lung cancer treated with chemoradiotherapy.

The relationship between splenic volume and the outcome of chemoradiotherapy for lung cancer has rarely been studied or addressed. The purpose of our study was to investigate whether splenic volume was associated with prognosis in patients treated with chemoradiotherapy for advanced or locally advanced non-small cell lung cancer (NSCLC).A retrospective investigation was conducted. Finally, 202 patients met the criteria and were included in the study. All patients were divided into 2 groups according to the optimum cutoff value of splenic volume for overall survival (OS). The optimum cutoff value was identified by X-tile software, and the OS and disease-free survival (DFS) were compared between the 2 groups of patients. The impact of splenic volume and other clinical characteristics on OS and DFS was analyzed using the Kaplan-Meier method and Cox proportional hazards model. Clinical characteristics were compared using chi-square or Fisher exact tests.The median (range) of splenic volume was 156.03 (28.55-828.11) cm. The optimal cutoff value of splenic volume was 288.4 cm. For univariate analyses, high splenic volume was associated with decreased OS (P = .025) and DFS (P = .044). In multivariate analyses, splenic volume remained an independent predictor of OS as a binary dependent variable (P = .003).Excessive splenic volume was associated with decreased OS and DFS in patients with NSCLC treated with chemoradiotherapy. Splenic volume should be regarded as an independent prognostic factor for patients treated with chemoradiotherapy for advanced or locally advanced NSCLC.