Transcriptional dynamic changes in energy metabolism, protein synthesis and cell cycle regulation reveal the biological adaptation mechanisms of juvenile Acrossocheilus wenchowensis under acute temperature changes.

In recent years, frequent acute temperature changes have posed a serious threat to the physiology and survival of fish. This study utilized RNA-Seq technology to analyze the transcriptional dynamics in the muscle tissues of Acrossocheilus wenchowensis under various acute temperature conditions (16◦C, 20◦C, 24◦C, 28◦C and 32◦C). Through comprehensive analysis, we identified 11509 differentially expressed genes (DEGs), a gene set (profiles 19) that was significantly up-regulated with increasing temperature, and two weighted gene co-expression network analysis (WGCNA) modules that were significantly correlated with acute temperature changes. Furthermore, we identified 28 transcription factors that are pivotal in oxidative stress and energy metabolism under acute temperature changes. Our results showed that, compared to the control group (24°C), KEGG functional enrichment analysis revealed significant enrichment of DEGs in the cell cycle, DNA replication, and p53 signaling pathway, with an overall trend of suppressed expression. This indicates that maintaining cell stability and reducing cell damage is an effective adaptive mechanism for A. wenchowensis to cope with acute temperature changes. Through STEM analysis and the black WGCNA module associated with high-temperature stress, we identified significant up-regulation of pathways and hub genes related to energy metabolism including oxidative phosphorylation, TCA cycle, purine metabolism, and glutathione metabolism, as well as the central roles of signal transduction pathways such as MAPK signaling pathway and AMPK signaling pathway, which synergistically regulate energy production. Under acute low-temperature stress, the turquoise WGCNA module highlighted significant up-regulation of hub genes associated with Ribosomal and Spliceosomal pathways related to protein synthesis and processing, as well as activation of calcium signaling pathways, which plays an important role in maintaining cellular function during low-temperature adaptation. These findings provide a critical theoretical and molecular basis for the adaptation of eurythermal fish to rapid temperature changes.

Pharmacokinetics, withdrawal period and risk assessment of enrofloxacin in the northern snakehead (Channa argus) following bath administration.

Enrofloxacin (ENR) is widely used in aquaculture practice, but little is known about its pharmacokinetic, withdrawal period and dietary risk in fish via bath administration. The purpose of this study was to provide data support for the use of ENR bath therapy in the northern snakehead (Channa argus). The pilot study was carried out to evaluate the therapy concentrations of ENR in northern snakehead with immersion concentrations ranged from 5 to 40 mg/L for 6 h. Based on results of the pilot study, an ENR immersion concentration of 20 mg/L was used for the formal experiment. At this dose, the peak concentrations of ENR in plasma, muscle plus skin, liver and kidney were 4.85, 4.55, 3.87 and 7.42 µg/mL (or g), respectively. According to the AUC0-∞ values, the distribution of ENR in northern snakehead followed the order of kidney > plasma > liver > muscle + skin. The elimination of ENR in northern snakehead was very slow, the half-lives (T1/2λz ) were up to 90.31, 85.5, 104.56 and 120.9 h in plasma, muscle plus skin, liver and kidney, respectively. Ciprofloxacin (CIP) was not detected in any samples in the pilot study and was only occasionally detected in muscle plus skin and liver samples in formal experiment. Based on the calculated PK/PD index AUC/MIC and Cmax /MIC, the current bath treatment regimen will have a good therapeutic effect on infections caused by bacteria with MIC below 0.6 µg/mL. The dietary risk assessment suggested that there was a dietary risk (Hazard Quotients > 10%) until day 6 after bath treatment. It is mandatory for ENR to maintain a withdrawal period of at least 450°C-day in northern snakehead after bath treatment ceased.

Perfluorooctanoic Acid (PFOA) Exposure Compromises Fertility by Affecting Ovarian and Oocyte Development.

PFOA, a newly emerging persistent organic pollutant, is widely present in various environmental media. Previous reports have proved that PFOA exposure can accumulate in the ovary and lead to reproductive toxicity in pregnant mice. However, the potential mechanism of PFOA exposure on fertility remains unclear. In this study, we explore how PFOA compromises fertility in the zebrafish. The data show that PFOA (100 mg/L for 15 days) exposure significantly impaired fertilization and hatching capability. Based on tissue sections, we found that PFOA exposure led to ovarian damage and a decrease in the percentage of mature oocytes. Moreover, through in vitro incubation, we determined that PFOA inhibits oocyte development. We also sequenced the transcriptome of the ovary of female zebrafish and a total of 284 overlapping DEGs were obtained. Functional enrichment analysis showed that 284 overlapping DEGs function mainly in complement and coagulation cascades signaling pathways. In addition, we identified genes that may be associated with immunity, such as LOC108191474 and ZGC:173837. We found that exposure to PFOA can cause an inflammatory response that can lead to ovarian damage and delayed oocyte development.

The complete mitochondrial genome of Neolissochilus stracheyi (Osteichthyes:Cyprinidae).

Neolissochilus stracheyi Day 1871 is a rare specie of fish inhabit clear forest streams and rivers. In order to discuss the phylogenetic position of N. stracheyi, the mitochondrial genome was obtained by sequencing. The genome was 16,587 bp in length with an accession number OM203155. The AT contents were 56.59%. The location and composition of genes are consistent with published Cyprinids containing 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 main non-coding regions. Sequence analysis showed that the mitochondrial genome of N. stracheyi has high sequence homology with other cyprinid fishes. Phylogenetic tree results showed that N. stracheyi is most closely related to Neolissochilus heterostomus. The mitochondrial sequence is of great significance for fish conservation, taxonomic status and resource exploitation.

The toxicokinetics and risk assessment of pyrethroids pesticide in tilapia (Oreochromis mossambicus) upon short-term water exposure.

Pyrethroids pesticides (PPs) are the widely adopted synthetic pesticides for agriculture and fishery. The frequent use of these pesticides leads to the accumulation of residues in the freshwater environments in China, subsequently affecting aquatic organisms and ecosystems. However, there are few reports on the toxicological and risk assessment of aquaculture aquatic products. In this study, the uptake, depuration kinetics and potential risk to human health and ecology of fenpropathrin, cypermethrin, fenvalerate, and deltamethrin were assessed using tilapia. The results indicated that four PPs were readily accumulated by tilapia. The bioconcentration factors (BCF) of the PPs in plasma and muscle were between 71.3 and 2112.1 L/kg and 23.9-295.3 L/kg, respectively. The half-lives (t1/2) of muscle and plasma were 2.90-9.20 d and 2.57-8.15 d. The risks of PPs residues in the muscle of tilapia and exposed water were evaluated by hazard quotient (HQ) and risk quotient (RQ). Although PPs residues in tilapia had a low dietary risk to human health, the residues in the exposed water had a high ecological risk to fish, daphnia, and green algae. Therefore, assessing the PPs content in freshwater aquaculture and monitoring their dosages and frequencies are highly necessitated to avoid their adverse effect on the aquaculture environment.

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.

Distribution and risk assessment of hexachlorobutadiene, pentachloroanisole, and chlorobenzenes in sediment and wild fish from a region affected by industrial and agricultural activities in South China.

Hexachlorobutadiene, pentachloroanisole, and chlorobenzenes are regulated to control their release into the environment. There is little information regarding the distribution and risks of these pollutants in Chinese rivers. Therefore, we selected a prosperous agricultural and industrial region in South China as our study area and investigated the contamination profiles and risks of these pollutants in sediment and fish tissue samples. The results showed that, when compared with their levels in sediment, these lipophilic pollutants tended to accumulate in fish tissues in the following order: liver > brain > muscle. Some trichlorobenzene was found to be the result of reductive dechlorination of higher chlorinated benzenes. Hexachlorobutadiene and hexachlorobenzene could pose medium risks at certain sampling sites, but in general, almost no risk was found to the ecosystem. When the estimated daily human intakes of analytes through fish consumption were calculated for different age groups, the results suggested the analytes were unlikely to be a serious health concern for human. Our results could be used to update the existing data on the occurrence of these pollutants in the aquatic environment and to provide information for further pollution control by the local government.

2,4-DCP compromises the fertilization capacity of mouse oocytes.

2,4-DCP (2,4-dichlorophenol) is an environmental estrogen that is ubiquitously distributed in the environment and widely used to produce herbicides and pharmaceutical intermediates. Although 2,4-DCP is suspected to have endocrine disruption, the reproductive toxicity of 2,4-DCP in mammals has not been adequately assessed. In the present study, we examined the effect of 2,4-DCP on the fertility of mouse eggs. The data showed that oral administration of 2,4-DCP (180 mg/kg/day for 7 days) compromises the fertilization rate of mouse oocytes. To further analyze the mechanism by which 2,4-DCP decreases fertilization, the key regulators and events during fertilization of mouse eggs were investigated. We found that the dynamics of cortical granules (CGs) were disrupted by showing the redistribution of CG free domain in 2,4-DCP-administered oocytes. This abnormality perturbed the sperm binding site in the zona pellucida (ZP) and dramatically reduced the number of sperm binding to the ZP of 2,4-DCP-administered oocytes. In addition, the abundance of Juno, a sperm receptor on the egg membrane, was also decreased and its distribution was mislocated in 2,4-DCP-administered oocytes. Finally, we validated that the defects of fertilization participants and events caused by 2,4-DCP might be mediated by the increased level of reactive oxygen species-induced apoptosis of oocytes. Therefore, we demonstrate that 2,4-DCP compromises the fertilization ability of mouse oocytes via inducing oxidative stress.

Melatonin ameliorates the fertilization capacity of oocytes exposed to 17α-ethynylestradiol.

17α-ethynylestradiol (EE2), a synthetic hormone that derives from the natural hormone estradiol, has been reported to alter the sex determination, sexual maturity and secondary sexual characteristics of exposed organisms. However, the adverse effects of EE2 on the oocyte quality have not fully determined. Here, we found that EE2 exposure compromised the fertilization capacity of mouse oocytes, while treatment of melatonin remarkably elevated the fertilization rate. Specifically, we observed that EE2 exposure led to the abnormal distribution and premature exocytosis of ovastacin, leading to the reduced number of sperm binding to the EE2-exposed oocytes. In addition, we found that the abundance of Juno, the sperm receptor on the oocyte membrane, was also diminished, which might be another potential cause leading to the fertilization failure of EE2-exposed oocytes. Finally, we demonstrated that melatonin improved the fertilization ability of EE2-exposed oocytes through eliminating the excessive ROS and inhibiting apoptosis.

Contamination and risk profiles of triclosan and triclocarban in sediments from a less urbanized region in China.

Triclosan and triclocarban are priority environmental contaminants of increasing concern to environmental health. After application, the chemicals enter the aquatic environment and easily distribute in bed sediment due to their hydrophobicity, and thus pose potential ecological risks. This study investigated the distribution and risks of triclosan and triclocarban in the sediment environment of a less urbanized region in South China. The sampling sites with high levels of triclosan and triclocarban were found to locate in the tributaries. When compared to other monitoring results obtained from more densely populated regions, the residues of triclosan and triclocarban in the investigated region were low, suggesting that these two chemicals conservation in sediment is related to anthropic activities. The results of risk quotients showed that high risks to aquatic organisms were posed by triclosan residues in sediment, while the risks to benthic organisms were quite low. Triclocarban residues in sediment posed minimal to medium risks to aquatic and benthic organisms. In summary, using either of the calculation methods of risk quotients, medium risks posed by the antimicrobials can be found in certain sampling sites with low population densities. The results may be useful in the development of evidence-based policies for the government.

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.

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.

Investigation of the interaction between the fate of antibiotics in aquafarms and their level in the environment.

China is the largest freshwater aquaculture producer and antibiotics consumer in the world, and rivers in China are generally polluted by antibiotics. However, there is little information available regarding the linkage of antibiotics in aquaculture and the aquatic environment. Therefore, this study investigated the fate of antibiotics in several open water culture-based freshwater aquafarms, including integrated livestock/fish systems and non-integrated fish ponds, and explored the contamination profiles of antibiotics in the Beijiang River. Then the study tried to clarify the two-way interaction of antibiotics in aquaculture and the environment. The results showed that, when compared with the effluent from livestock farms and wastewater treatment plants, the contribution of antibiotics from non-integrated fish pond water without livestock sewage input was limited, while that of effluent from the integrated livestock/fish system was quite high. The total concentrations of antibiotics detected in the aquafarm source water were similar to those in the upper river water and generally higher than those in the corresponding fish pond water, implying that the occurrence of antibiotics in intensive aquafarms can mainly be attributed to the antibiotic residues in nearby river water. Overall, the results underscore the need to develop a sewage infrastructure for the treatment of effluent from integrated livestock/fish aquafarms, and suggest that open water culture-based fish farms should be located far from seriously contaminated sections of rivers.

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.

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.

Dynein promotes porcine oocyte meiotic progression by maintaining cytoskeletal structures and cortical granule arrangement.

Cytoplasmic dynein is a family of cytoskeletal motor proteins that move towards the minus-end of the microtubules to perform functions in a variety of mitotic processes such as cargo transport, organelle positioning, chromosome movement and centrosome assembly. However, its specific roles during mammalian oocyte meiosis have not been fully defined. Herein, we investigated the critical events during porcine oocyte meiotic maturation after inhibition of dynein by Ciliobrevin D treatment. We found that oocyte meiotic progression was arrested when inhibited of dynein by showing the poor expansion of cumulus cells and decreased rate of polar body extrusion. Meanwhile, the spindle assembly and chromosome alignment were disrupted, accompanied by the reduced level of acetylated α-tubulin, indicative of weakened microtubule stability. Defective actin polymerization on the plasma membrane was also observed in dynein-inhibited oocytes. In addition, inhibition of dynein caused the abnormal distribution of cortical granules and precocious exocytosis of ovastacin, a cortical granule component, which predicts that ZP2, the sperm binding site in the zona pellucida, might be prematurely cleaved in the unfertilized dynein-inhibited oocytes, potentially leading to the fertilization failure. Collectively, our findings reveal that dynein plays a part in porcine oocyte meiotic progression by regulating the cytoskeleton dynamics including microtubule stability, spindle assembly, chromosome alignment and actin polymerization. We also find that dynein mediates the normal cortical granule distribution and exocytosis timing of ovastacin in unfertilized eggs which are the essential for the successful fertilization.

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.

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.

Smc1ß is required for activation of SAC during mouse oocyte meiosis.

Smc1ß is a meiosis-specific cohesin subunit that is essential for sister chromatid cohesion and DNA recombination. Previous studies have shown that Smc1ß-deficient mice in both sexes are sterile. Ablation of Smc1ß during male meiosis leads to the blockage of spermatogenesis in pachytene stage, and ablation of Smc1ß during female meiosis generates a highly error-prone oocyte although it could develop to metaphase II stage. However, the underlying mechanisms regarding how Smc1ß maintains the correct meiotic progression in mouse oocytes have not been clearly defined. Here, we find that GFP-fused Smc1ß is expressed and localized to the chromosomes from GV to MII stages during mouse oocyte meiotic maturation. Knockdown of Smc1ß by microinjection of gene-specific morpholino causes the impaired spindle apparatus and chromosome alignment which are highly correlated with the defective kinetochore-microtubule attachments, consequently resulting in a prominently higher incidence of aneuploid eggs. In addition, the premature extrusion of polar bodies and escape of metaphase I arrest induced by low dose of nocodazole treatment in Smc1ß-depleted oocytes indicates that Smc1ß is essential for activation of spindle assembly checkpoint (SAC) activity. Collectively, we identify a novel function of Smc1ß as a SAC participant beyond its role in chromosome cohesion during mouse oocyte meiosis.

Stag3 regulates microtubule stability to maintain euploidy during mouse oocyte meiotic maturation.

Stag3, a meiosis-specific subunit of cohesin complex, has been demonstrated to function in both male and female reproductive systems in mammals. However, its roles during oocyte meiotic maturation have not been fully defined. In the present study, we report that Stag3 uniquely accumulates on the spindle apparatus and colocalizes with microtubule fibers during mouse oocyte meiotic maturation. Depletion of Stag3 by gene-targeting morpholino disrupts normal spindle assembly and chromosome alignment in oocytes. We also find that depletion of Stag3 reduces the acetylated level of tubulin and microtubule resistance to microtubule depolymerizing drug, suggesting that Stag3 is required for microtubule stability. Consistent with these observations, kinetochore-microtubule attachment, an important mechanism controlling chromosome alignment, is severely impaired in Stag3-depleted oocytes, resultantly causing the significantly increased incidence of aneuploid eggs. Collectively, our data reveal that Stag3 is a novel regulator of microtubule dynamics to ensure euploidy during moue oocyte meiotic maturation.