Botrytis cinerea mediated cell wall degradation accelerates spike stalk browning in Munage grape.

Munage grape (Vitis vinifera L. cv. Munage.) is a unique cultivar in southern Xinjiang, China. Spike stalk browning in this species has becomes more common in recent years, negatively impacting the shelf life, and causing severe economic losses during storage. This study investigated the changes in metabolisms of cell wall by Botrytis cinerea infection in association with spike stalk browning. Morphological and physiological observations showed that preharvest B. cinerea infection accelerates the spike stalk browning during storage in Munage grapes by promoting cell wall degradation. Accordingly, the cell structures in infected spike stalk showed severe collapse, while the cell structures in uninfected spike stalk remained relatively complete. Furthermore, the contents of CDTA-soluble pectin (CSP), Na2 CO3 -soluble pectin (NSP), cellulose, and hemicellulose were reduced, while the water-soluble pectin (WSP) content was increased during infection. In addition, the activities of polygalacturonase (PG), pectin methylesterase (PME), beta-galactosidase (ß-Gal), and cellulase (Cx) were highly promoted by B. cinerea. Correspondingly, the expression levels of VvPG were markedly upregulated after inoculation and played a major role in cell wall degradation. Additionally, the spike stalk inoculated by B. cinerea showed higher activities of PPO and POD, and content of total phenolics. These results contribute to elucidating the relationship between cell wall degradation induced by B. cinerea during spike stalk browning and provide a basis for future research on improving the ability of the host cell wall to resist degrading enzymes. PRACTICAL APPLICATIONS: Botrytis cinerea is the main fungal pathogen causing the gray mold of grapes. It usually enters the tissue early in crop development, has a long incubation period, and rapidly infects the tissue when the environment is favorable and the host physiology changes. Gray mold has been reported as one of the major postharvest diseases of grapes. However, there are relatively few reports on the pathways through which B. cinerea causes the browning of grape stalks. Controlling browning caused by B. cinerea may require clarification of the physiological and molecular mechanisms by which browning occurs. The elucidation of the role of B. cinerea in causing browning of grape stalks through the cell wall degradation pathway will help to provide scientific basis for further controlling browning, maintaining freshness of stalks, developing biological agents to prevent browning, improving grape quality, and extending storage period.

Melatonin protects in vitro matured porcine oocytes from toxicity of Aflatoxin B1.

Aflatoxin B1 (AFB1) is a major food and feed contaminant that threaten public health. Previous studies indicate that AFB1 exposure disrupted oocyte maturation. However, an effective and feasible method is unavailable for protecting oocytes against toxicity of AFB1. In the present study, using in vitro matured porcine oocytes and parthenogenetic embryos as model, we confirmed that AFB1 exposure during in vitro oocyte maturation (IVM) significantly impaired both nuclear and cytoplasmic maturation in a dose- and time-dependent manner. The different concentrations of melatonin were also tested for their protective effects on oocytes against the AFB1-induced toxicity. Our results showed that supplementation of a relative high concentration of melatonin (10-3 mol/L) during IVM efficiently reversed the impaired development rate and blastocyst quality, to the levels comparable to those of the control group. Further analysis indicated that melatonin application efficiently alleviated reactive oxygen species accumulation and initiation of apoptosis induced by AFB1 exposure. In addition, disrupted GSH/GPX system, as well as inhibited mitochondrial DNA (mtDNA) replication and mitochondrial biogenesis in AFB1-treated oocytes, can be notably reversed by melatonin application. Furthermore, cumulus cells may be important in mediating the toxicity of AFB1 to oocytes, and the metabolism of AFB1 in cumulus cells can be depressed by melatonin. To the best of our knowledge, this is the first report to confirm that melatonin application can efficiently protect oocytes from AFB1-induced toxicity. Our study provides a promising and practical strategy for alleviating or reversing AFB1-induced female reproductive toxicity in both clinical treatment and domestic reproductive management.

Impaired imprinted X chromosome inactivation is responsible for the skewed sex ratio following in vitro fertilization.

Dynamic epigenetic reprogramming occurs during normal embryonic development at the preimplantation stage. Erroneous epigenetic modifications due to environmental perturbations such as manipulation and culture of embryos during in vitro fertilization (IVF) are linked to various short- or long-term consequences. Among these, the skewed sex ratio, an indicator of reproductive hazards, was reported in bovine and porcine embryos and even human IVF newborns. However, since the first case of sex skewing reported in 1991, the underlying mechanisms remain unclear. We reported herein that sex ratio is skewed in mouse IVF offspring, and this was a result of female-biased peri-implantation developmental defects that were originated from impaired imprinted X chromosome inactivation (iXCI) through reduced ring finger protein 12 (Rnf12)/X-inactive specific transcript (Xist) expression. Compensation of impaired iXCI by overexpression of Rnf12 to up-regulate Xist significantly rescued female-biased developmental defects and corrected sex ratio in IVF offspring. Moreover, supplementation of an epigenetic modulator retinoic acid in embryo culture medium up-regulated Rnf12/Xist expression, improved iXCI, and successfully redeemed the skewed sex ratio to nearly 50% in mouse IVF offspring. Thus, our data show that iXCI is one of the major epigenetic barriers for the developmental competence of female embryos during preimplantation stage, and targeting erroneous epigenetic modifications may provide a potential approach for preventing IVF-associated complications.

Actin Disorganization Plays a Vital Role in Impaired Embryonic Development of In Vitro-Produced Mouse Preimplantation Embryos.

Assisted reproductive technology (ART) is being increasingly applied to overcome infertility. However, the in vitro production process, the main procedure of ART, can lead to aberrant embryonic development and health-related problems in offspring. Understanding the mechanisms underlying the ART-induced side effects is important to improve the ART process. In this study, we carried out comparative transcriptome profiling between in vivo- (IVO) and in vitro- produced (IVP) mouse blastocysts. Our results suggested that aberrant actin organization might be a major factor contributing to the impaired development of IVP embryos. To test this, we examined the effect of actin disorganization on the development of IVP preimplantation embryos. Specific disruption of actin organization by cytochalasin B (CB) indicated that well-organized actin is essential for in vitro embryonic development. Supplementing the culture medium with 10(-9) M melatonin, a cytoskeletal modulator in adult somatic cells, significantly reversed the disrupted expression patterns of genes related to actin organization, including Arhgef2, Bcl2, Coro2b, Flnc, and Palld. Immunofluorescence analysis showed that melatonin treatment of IVP embryos significantly improved the distribution and organization of actin filaments (F-actin) from the 8-cell stage onwards. More importantly, we found that melatonin alleviated the CB-mediated aberrant F-actin distribution and organization and rescued CB-induced impaired embryonic development. This is the first study to indicate that actin disorganization is implicated in impaired development of IVP embryos during the preimplantation stage. We also demonstrated that improving actin organization is a promising strategy to optimize existing IVP systems.

Treatment of porcine donor cells and reconstructed embryos with the antioxidant melatonin enhances cloning efficiency.

This study was conducted to investigate the effect of melatonin during the culture of donor cells and cloned embryos on the in vitro developmental competence and quality of cloned porcine embryos. At concentrations of 10(-6 )M or 10(-8) M, melatonin significantly enhanced the proliferation of porcine fetal fibroblasts (PFFs), and the blastocyst rate was significantly increased in the 10(-10) M melatonin-treated donor cell group. Cloned embryo development was also improved in embryo culture medium that was supplemented with 10(-9) M or 10(-12) M melatonin. When both donor cells and cloned embryos were treated with melatonin, the cleavage rate and total cell number of blastocysts were not significantly affected; however, the blastocyst rate was increased significantly (20.0% versus 11.7%). TUNEL assays showed that combined melatonin treatment reduced the rate of apoptotic nuclei (3.6% versus 6.1%). Gene expression analysis of the apoptosis-related genes BAX, BCL2L1, and p53 showed that the expression of BCL2L1 was significantly elevated 2.7-fold relative to the control group, while the expression of BAX and p53 was significantly decreased by 3.7-fold and 23.2-fold, respectively. In addition, we detected the expression of two melatonin receptors (MT1 and MT2) in PFFs but not in porcine cloned embryos. We conclude that exogenous melatonin enhances the development of porcine cloned embryos and improves embryo quality by inhibiting p53-mediated apoptotic pathway. The proliferation of PFFs may be mediated by receptor binding, but the beneficial effects of melatonin on embryonic development may be receptor-independent, possibly through melatonin's ability to directly scavenge free radicals.

Melatonin improves the reprogramming efficiency of murine-induced pluripotent stem cells using a secondary inducible system.

This study focused on the effect of melatonin on reprogramming with specific regard to the generation of induced pluripotent stem cells (iPSCs). Here, a secondary inducible system, which is more accurate and suitable for studying the involvement of chemicals in reprogramming efficiency, was used to evaluate the effect of melatonin on mouse iPSC generation. Secondary fibroblasts collected from all-iPSC mice through tetraploid complementation were cultured in induction medium supplemented with melatonin at different concentrations (0, 10(-6), 10(-7), 10(-8), 10(-9), or 10(-10 )m) or with vitamin C (50 µg/mL) as a positive control. Compared with untreated group (0.22 ± 0.04% efficiency), 10(-8) (0.81 ± 0.04%), and 10(-9 )m (0.83 ± 0.08%) melatonin supplementation significantly improved reprogramming efficiency (P < 0.05). Moreover, we verified that the iPSCs induced by melatonin treatment (MiPSCs) had the same characteristics as typical embryonic stem cells (ESCs), including expression of the pluripotency markers Oct4, Sox2, and Nanog, the ability to form teratomas and all three germ layers of the embryo, as well as produce chimeric mice with contribution to the germ line. Interestingly, only the melatonin receptor MT2 was detected in secondary fibroblasts, while MiPSCs and ESCs expressed MT1 and MT2 receptors. Furthermore, during the early stage of reprogramming, expression of the apoptosis-related genes p53 and p21 was lower in the group treated with 10(-9) m melatonin compared with the untreated controls. In conclusion, melatonin supplementation enhances the efficiency of murine iPSC generation. These beneficial effects may be associated with inhibition of the p53-mediated apoptotic pathway.

Effect of antioxidant supplementation on function and fertility of sex-sorted boar spermatozoa.

The goal of this study was to investigate the effects of antioxidant supplementation on the quality of flow cytometrically-sorted boar spermatozoa. The effects of ascorbic acid-2-glucoside (AA-2G) on the sex-sorting process were evaluated using a variety of concentrations. The effects of different antioxidants (AA-2G, l-glutathione, and vitamin E) on the viability and lifespan of boar spermatozoa were also compared during sorting. Furthermore, the effect of AA-2G on acrosome intactness, the capacitation ability of sorted boar spermatozoa and pregnancy efficiency after artificial insemination (AI) at different sorting-to-insemination intervals were examined. Greater (P<0.05) percentages of motile spermatozoa and acrosome intactness and longer storage time periods were observed in the AA-2G-supplemented group when compared with the other antioxidant-supplemented or control groups. At an AA-2G concentration of 0.068 mg/mL, the motility characteristics (i.e., straightness (STR), velocity according to the average path (VAP), and amplitude of the sperm head lateral displacement (ALH)) of the sex-sorted boar spermatozoa were greater (P<0.05) than in those treated with other AA-2G concentrations. The capacitation rate of boar spermatozoa in the AA-2G-supplemented group was less (P<0.05) than that in the control group. After sorting-to-insemination interval of 10h, the pregnancy rates after AI with boar spermatozoa for the AA-2G-supplemented group were 59.25%, while the control group remains no sufficient quality semen. This study demonstrates that AA-2G supplementation can improve the quality of flow cytometrically sorted boar spermatozoa and that the optimal concentration of AA-2G for sorting is 0.068 mg/mL.