Qiao-Chu Wang

Deoxynivalenol exposure induces autophagy/apoptosis and epigenetic modification changes during porcine oocyte maturation

Deoxynivalenol (DON) is a widespread trichothecene mycotoxin which contaminates agricultural staples and elicits a complex spectrum of toxic effects on humans and animals. It has been shown that DON impairs oocyte maturation, reproductive function and causes abnormal fetal development in mammals; however, the mechanisms remain unclear. In the present study, we investigate the possible reasons of the toxic effects of DON on porcine oocytes. Our results showed that DON significantly inhibited porcine oocyte maturation and disrupted meiotic spindle by reducing p-MAPK protein level, which caused retardation of cell cycle progression. In addition, up-regulated LC3 protein expression and aberrant Lamp2, LC3 and mTOR mRNA levels were observed with DON exposure, together with Annexin V-FITC staining assay analysis, these results indicated that DON treatment induced autophagy/apoptosis in porcine oocytes. We also showed that DON exposure increased DNA methylation level in porcine oocytes through altering DNMT3A mRNA levels. Histone methylation levels were also changed showing with increased H3K27me3 and H3K4me2 protein levels, and mRNA levels of their relative methyltransferase genes, indicating that epigenetic modifications were affected. Taken together, our results suggested that DON exposure reduced porcine oocytes maturation capability through affecting cytoskeletal dynamics, cell cycle, autophagy/apoptosis and epigenetic modifications.

Aflatoxin B1 is toxic to porcine oocyte maturation

As a toxic secondary metabolite of Aspergillus species, Aflatoxin B1 (AFB1) is a major food and feed contaminant in tropical and sub-tropical regions with high temperature and humidity. It has been reported to be toxic to the female reproductive system in laboratory and domestic animals. In the present study, the influence of acute exposure to AFB1 (10 and 50 μM, 44h) on porcine oocyte maturation and its possible mechanism were investigated. The maturation rates of oocytes decreased significantly in the presence of 50 μM of AFB1. Cell cycle analysis showed that most oocytes were arrested at germinal vesicle breakdown or meosis I stage. However, actin assembly, spindle structure and chromosome alignment were not disrupted after exposure to 50 μM AFB1. Further study showed that DNA methylation levels increased in treated oocytes (50 μM). Histone methylation levels were also analysed after treatment (50 μM): H3K27me3 and H3K4me2 levels decreased, whereas H3K9me3 level increased, indicating that epigenetic modification was affected. AFB1 treatment (50 μM) also induced oxidative stress and further led to autophagy, as shown by accumulation of reactive oxygen species, up-regulated LC3 protein expression and increased mRNA levels of ATG3, ATG5 and ATG7. Annexin V-FITC staining assay revealed that AFB1 treatment (50 μM) resulted in oocyte early apoptosis, which was confirmed by increased Bak, Bax, Bcl-xl mRNA levels. Collectively, our results suggest that AFB1 disrupts porcine oocyte maturation through changing epigenetic modifications as well as inducing oxidative stress, excessive autophagy and apoptosis.

Acrylamide toxic effects on mouse oocyte quality and fertility in vivo

Acrylamide is an industrial chemical that has attracted considerable attention due to its presumed carcinogenic, neurotoxic, and cytotoxic effects. In this study we investigated possible acrylamide reproductive toxic effects in female mice. Mice were fed an acrylamide-containing diet for 6 weeks. Our results showed the following effects of an acrylamide-containing diet. (1) Ovary weights were reduced in acrylamide-treated mice and oocyte developmental competence was also reduced, as shown by reduced GVBD and polar body extrusion rates. (2) Acrylamide feeding resulted in aberrant oocyte cytoskeletons, as shown by an increased abnormal spindle rate and confirmed by disrupted γ-tubulin and p-MAPK localization. (3) Acrylamide feeding resulted in oxidative stress and oocyte early stage apoptosis, as shown by increased ROS levels and p-MAPK expression. (4) Fluorescence intensity analysis showed that DNA methylation levels were reduced in acrylamide-treated oocytes and histone methylation levels were also altered, as H3K9me2, H3K9me3, H3K4me2, and H3K27me3 levels were reduced after acrylamide treatment. (5) After acrylamide feeding, the litter sizes of acrylamide-treated mice were significantly smaller compared to thus of control mice. Thus, our results indicated that acrylamide might affect oocyte quality through its effects on cytoskeletal integrity, ROS generation, apoptosis induction, and epigenetic modifications.

MKlp2 inhibitior paprotrain affects polar body extrusion during mouse oocyte maturation

BackgroundMammalian oocyte meiotic maturation involves a number of important processes, including spindle assembly and migration, cortical reorganization and polar body extrusion. Numerous proteins contribute to these processes, but it is unknown whether MKlp2 (mitotic kinesin-like protein 2; also called KIF20A), a microtubule-associated protein that regulates cytokinesis during mitosis, is involved in oocyte maturation.MethodsConfocal microscopy, time lapse microscopy, inhibitor treatment were adopted to examine the roles of MKlp2 in mouse oocyte.ResultsImmunostaining results showed that MKlp2 localized to oocyte microtubules. Time-lapse microscopy showed that disrupting MKlp2 expression with paprotrain, a specific inhibitor of MKlp2, resulted in polar body extrusion failure. This could be rescued after rescuing oocytes from paprotrain in fresh medium. Cell cycle analysis showed that most oocytes were arrested at metaphase I or telophase I. However, oocyte spindle structure and chromosome alignment were not disrupted after the inhibition of MKlp2 by paprotrain.ConclusionsThis study demonstrated that MKlp2 is crucial for oocyte maturation by regulating polar body extrusion.

Nucleation promoting factors regulate the expression and localization of Arp2/3 complex during meiosis of mouse oocytes.

The actin nucleation factor Arp2/3 complex is a main regulator of actin assembly and is involved in multiple processes like cell migration and adhesion, endocytosis, and the establishment of cell polarity in mitosis. Our previous work showed that the Arp2/3 complex was involved in the actin-mediated mammalian oocyte asymmetric division. However, the regulatory mechanisms and signaling pathway of Arp2/3 complex in meiosis is still unclear. In the present work, we identified that the nucleation promoting factors (NPFs) JMY and WAVE2 were necessary for the expression and localization of Arp2/3 complex in mouse oocytes. RNAi of both caused the degradation of actin cap intensity, indicating the roles of NPFs in the formation of actin cap. Moreover, JMY and WAVE2 RNAi decreased the expression of ARP2, a key component of Arp2/3 complex. However, knock down of Arp2/3 complex by Arpc2 and Arpc3 siRNA microinjection did not affect the expression and localization of JMY and WAVE2. Our results indicate that the NPFs, JMY and WAVE2, are upstream regulators of Arp2/3 complex in mammalian oocyte asymmetric division.

Trace element concentrations and distributions in the main body tissues and the net requirements for maintenance and growth of Dorper × Hu lambs

A comparative slaughter trial was conducted to estimate the trace element concentrations and distributions in the main body tissues and the net requirements for maintenance and growth of Dorper × Hu crossbred lambs. Thirty-five lambs of each gender (19.2 ± 0.36 kg initial BW) were used. Seven lambs of each gender were randomly chosen and slaughtered at approximately 20 kg BW as the baseline group for measuring initial body composition. Another 7 lambs of each gender were also randomly chosen and offered a pelleted mixed diet for ad libitum intake and slaughtered at approximately 28 kg BW. The remaining 21 sheep of each gender were randomly divided into 3 groups with 7 sheep each and assigned to ad libitum or 40 or 70% of ad libitum intake of a pelleted mixed diet (42:58 concentrate:roughage, DM basis). The 3 groups of each gender were slaughtered when the sheep fed ad libitum reached approximately 35 kg BW. Empty body (head + feet, hide, viscera + blood, and carcass) trace element contents were determined after slaughter. The results showed that the trace elements were mainly distributed in viscera (blood included), except for Zn, which was mainly distributed in the muscle and bone tissues. The net requirements were calculated using the comparative slaughter technique. For males and females, the daily net trace element requirements for maintenance were 356.1 and 164.1 μg Fe, 4.3 and 3.4 μg Mn, 42.0 and 29.8 μg Cu, and 83.5 and 102.0 μg Zn per kilogram empty body weight (EBW), respectively. Net requirements for growth decreased from 65.67 to 57.27 mg Fe, 0.35 to 0.25 mg Mn, and 3.45 to 2.82 mg Cu and increased from 26.36 to 26.65 mg Zn per kilogram EBW gain (EBWG) for males. Net requirements for growth decreased from 30.66 to 22.14 mg Fe, 0.43 to 0.32 mg Mn, 2.86 to 2.18 mg Cu, and 27.71 to 25.83 mg Zn per kilogram EBWG for females from 20 to 35 kg BW. This study indicated that the net trace element requirements for Dorper × Hu crossbred lambs may be different from those of purebred or other genotypes, and more data are needed for sheep in general.

Dynamin 2 regulates actin-mediated spindle migration in mouse oocytes

During meiosis, a bipolar spindle forms in the central cytoplasm of an oocyte and then moves to the cortex to extrude the first polar body. This is dependent on the regulation of actin and actin‐related molecules. Dynamin 2, a large guanosine triphosphatases (GTPase) known to regulate clathrin‐mediated endocytosis, is involved in actin recruitment and actin‐based vesicle mobility. In this study, we investigated the role of Dynamin 2 in oocyte meiosis.

RhoA-mediated MLC2 regulates actin dynamics for cytokinesis in meiosis

ABSTRACT During oocyte meiosis, the bipolar spindle forms in the central cytoplasm and then migrates to the cortex. Subsequently, the oocyte extrudes the polar body through two successive asymmetric divisions, which are regulated primarily by actin filaments. Myosin light chain2 (MLC2) phosphorylation plays pivotal roles in smooth muscle contraction, stress fiber formation, cell motility and cytokinesis. However, whether MLC2 phosphorylation participates in the oocyte polarization and asymmetric division has not been clarified. The present study investigated the expression and functions of MLC2 during mouse oocyte meiosis. Our result showed that p-MLC2 was localized in the oocyte cortex, with a thickened cap above the chromosomes. Meanwhile, p-MLC2 was also localized in the poles of spindle. Disruption of MLC2 activity by MLC2 knock down (KD) caused the failure of polar body extrusion. Immunofluorescent staining showed that a large proportion of oocytes arrested in telophase stage and failed to undergo cytokinesis after culturing for 12 hours. In the meantime, actin filament staining at oocyte membrane and cytoplasm were reduced in MLC2 KD oocytes. Finally, we found that the phosphorylation of MLC2 protein levels was decreased after disruption of RhoA activity. Above all, our data indicated that the RhoA-mediated MLC2 regulates the actin organization for cytokinesis during mouse oocyte maturation.

Role of nucleation-promoting factors in mouse early embryo development.

During mitosis nucleation-promoting factors (NPFs) bind to the Arp2/3 complex and activate actin assembly. JMY and WAVE2 are two critical members of the NPFs. Previous studies have demonstrated that NPFs promote multiple processes such as cell migration and cytokinesis. However, the role of NPFs in development of mammalian embryos is still unknown. Results of the present study show that the NPFs JMY and WAVE2 are critical for cytokinesis during development of mouse embryos. Both JMY and WAVE2 are expressed in mouse embryos. After injection of JMY or WAVE2 siRNA, all embryos failed to develop to the morula or blastocyst stages. Moreover, using fluorescence intensity analysis, we found that the expression of actin decreased, and multiple nuclei were observed within a single cell indicating that NPFs-induced actin reduction caused the failure of cell division. In addition, injection of JMY and WAVE2 siRNA also caused ARP2 degradation, indicating that involvement of NPFs in development of mouse embryos is mainly through regulation of ARP2/3-induced actin assembly. Taken together, these data suggested that WAVE2 and JMY are involved in development of mouse embryos, and their regulation may be through a NPFs-Arp2/3-actin pathway.

Involvement of Dynamin 2 in actin-based polar-body extrusion during porcine oocyte maturation.

Mammalian oocyte meiotic maturation involves a unique asymmetric division, but the regulatory mechanisms and signaling pathways involved are poorly understood. Dynamins are ubiquitous eukaryotic GTPases involved in membrane trafficking and actin dynamics, whose roles in mammalian oocyte maturation have not been determined. In this study, we used porcine oocytes to show that Dynamin 2 accumulated at the meiotic spindle and in the cortex of oocytes, with a distribution similar to that of actin. Inhibiting Dynamin 2 activity in porcine oocytes with the specific inhibitor dynasore resulted in failed polar‐body extrusion. This phenotype may have been due to aberrant actin distribution and/or spindle positioning as inhibitor treatment disrupted the formation of the actin cap and cortical granule‐free domain, which negatively impacted spindle positioning. Moreover, the distribution of ARP2, a key actin‐nucleation factor, was severely reduced in the cortex after dynasore treatment. Thus, our results suggest that Dynamin 2 possibly regulates porcine oocyte maturation through its effects on actin‐mediated spindle positioning and cytokinesis, and that this may depend on regulating ARP2 localization. Mol. Reprod. Dev. 81: 725–734, 2014.