-
[show abstract]
[hide abstract]
ABSTRACT: MicroRNAs (miRNAs) are a class of highly conserved small non-coding RNA molecules that play a pivotal role in several cellular functions. In this study, miRNA and messenger RNA (mRNA) profiles were examined by Illumina microarray in mouse embryonic stem cells (ESCs) derived from parthenogenetic, androgenetic, and fertilized blastocysts. The global analysis of miRNA-mRNA target pairs provided insight into the role of miRNAs in gene expression. Results showed that a total of 125 miRNAs and 2394 mRNAs were differentially expressed between androgenetic ESCs (aESCs) and fertilized ESCs (fESCs), a total of 42 miRNAs and 87 mRNAs were differentially expressed between parthenogenetic ESCs (pESCs) and fESCs, and a total of 99 miRNAs and 1788 mRNAs were differentially expressed between aESCs and pESCs. In addition, a total of 575, 5 and 376 miRNA-mRNA target pairs were observed in aESCs vs. fESCs, pESCs vs. fESCs, and aESCs vs. pESCs, respectively. Furthermore, 15 known imprinted genes and 16 putative uniparentally expressed miRNAs with high expression levels were confirmed by both microarray and real-time RT-PCR. Finally, transfection of miRNA inhibitors was performed to validate the regulatory relationship between putative maternally expressed miRNAs and target mRNAs. Inhibition of miR-880 increased the expression of Peg3, Dyrk1b, and Prrg2 mRNA, inhibition of miR-363 increased the expression of Nfat5 and Soat1 mRNA, and inhibition of miR-883b-5p increased Nfat5, Tacstd2, and Ppapdc1 mRNA. These results warrant a functional study to fully understand the underlying regulation of genomic imprinting in early embryo development.
Journal of Genetics and Genomics 04/2013; 40(4):189-200. · 1.88 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Microscopy and Microanalysis 04/2013; · 3.01 Impact Factor
-
Yong-Nan Xu,
Sang-Jun Uhm,
Bon-Chul Koo,
Mo-Sun Kwon,
Ji-Yeol Roh,
Jung-Seok Yang,
Hyun-Yong Choi,
Young-Tae Heo, Xiang-Shun Cui,
Joon-Ho Yoon,
Dae-Hwan Ko,
Teoan Kim,
Nam-Hyung Kim
[show abstract]
[hide abstract]
ABSTRACT: The potential benefits of generating and using transgenic cattle range from improvements in agriculture to the production of large quantities of pharmaceutically relevant proteins. Previous studies have attempted to produce transgenic cattle and other livestock by pronuclear injection and somatic cell nuclear transfer, but these approaches have been largely ineffective; however, a third approach, lentivirus-mediated transgenesis, has successfully produced transgenic livestock. In this study, we generated transgenic (TG) Korean native cattle using perivitelline space injection of viral vectors, which expressed enhanced green fluorescent protein (EGFP) systemically. Two different types of lentiviral vectors derived from feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV) carrying EGFP were injected into the perivitelline space of MII oocytes. EGFP expression at 8-cell stage was significantly higher in the FIV group compared to the HIV group (47.5%±2.2% v.s. 22.9%±2.9%). Eight-cell embryos that expressed EGFP were cultured into blastocysts and then transferred into 40 heifers. Ten heifers were successfully impregnated and delivered 10 healthy calves. All of these calves expressed EGFP as detected by in vivo imaging, PCR and Southern blotting. In addition, we established an EGFP-expressing cell line from TG calves, which was followed by nuclear transfer (NT). Recloned 8-cell embryos also expressed EGFP, and there were no differences in the rates of fusion, cleavage and development between cells derived from TG and non-TG calves, which were subsequently used for NT. These results illustrate that FIV-based lentiviruses are useful for the production of TG cattle. Moreover, our established EGFP cell line can be used for additional studies that involve induced pluripotent stem cells.
Journal of Genetics and Genomics 01/2013; 40(1):37-43. · 1.88 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Mycotoxins which mainly consist of Aflatoxin (AF), Zearalenone (ZEN) and Deoxynivalenol (DON) are commonly found in many food commodities. Although each component has been shown to cause liver toxicity and oxidative stress in several species, there is no evidence regarding the effect of naturally contained multiple mycotoxins on tissue toxicity and oxidative stress in vivo. In the present study, mycotoxins-contaminated maize (AF 597 µg/kg, ZEN 729 µg/kg, DON 3.1 mg/kg maize) was incorporated into the diet at three different doses (0, 5 and 20%) to feed the mice, and blood and tissue samples were collected to examine the oxidative stress related indexes. The results showed that the indexes of liver, kidney and spleen were all increased and the liver and kidney morphologies changed in the mycotoxin-treated mice. Also, the treatment resulted in the elevated glutathione peroxidase (GPx) activity and malondialdehyde (MDA) level in the serum and liver, indicating the presence of the oxidative stress. Moreover, the decrease of catalase (CAT) activity in the serum, liver and kidney as well as superoxide dismutase (SOD) activity in the liver and kidney tissue further confirmed the occurrence of oxidative stress. In conclusion, our data indicate that the naturally contained mycotoxins are toxic in vivo and able to induce the oxidant stress in the mouse.
PLoS ONE 01/2013; 8(3):e60374. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The effect of Doxorubicin, a widely used chemotherapeutic agent, on early mouse embryonic development has not been previously characterized.
Expression of apoptosis-related genes and poly(ADP-ribose) polymerase (PARP) family genes were assessed by real-time reverse transcription polymerase chain reaction (RT-PCR). Apoptosis in mouse blastocysts was tested using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Cleaved (c)-PARP was analyzed by western blot.
A 20 h exposure to doxorubicin caused rapid cytoplasmic fragmentation, DNA condensation and disruption of the cytoskeleton in mouse embryos. Doxorubicin altered the expression of genes involved in DNA repair and apoptosis and blocked early embryonic development, suggesting that doxorubicin affects DNA synthesis and repair. Furthermore, the effect of doxorubicin on early embryo development was determined by assessing the rates of development to different stages and an apoptosis index. Both assays confirmed that doxorubicin altered embryonic development. In conclusion, doxorubicin blocked pre-implantation development in early mouse embryos by altering apoptosis-related gene expression and inactivating DNA repair by PARP.
In vivo (Athens, Greece) 09/2012; 26(5):827-34. · 1.17 Impact Factor
-
Reproduction Fertility and Development 07/2012; · 2.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Autophagy, an essential process for cellular maintenance, cell viability, and development, is the bulk degradation of proteins and organelles. This study investigated the expression levels of autophagy-related genes and the effect of 3-methyladenine (3-MA, an autophagy inhibitor) or rapamycin (an autophagy inducer) on maternal gene degradation and apoptosis in porcine parthenotes developing in vitro. LC3, which is essential for the formation of autophagosomes, was widely expressed in porcine parthenotes. High levels of autophagy-related genes, Atg5, Beclin1 and Lc3 transcripts were expressed in the 1-cell (1C) stage and gradually decreased through the 2-cell (2C) to blastocyst stages. The mRNA expression of Gdf9, c-mos and cyclin B maintained high levels in 2C and 4-cell (4C) embryos treated with 3-MA compared with the control. The Bmp15 and cyclin B mRNA levels were significantly reduced in embryos treated with rapamycin compared with the control. These results suggest that autophagy influences the degradation of these maternal genes. Furthermore, 3-MA-treated embryos exhibited significantly reduced developmental rates, decreased total cell numbers and increased rates of apoptosis. Expression of Atg5, Beclin1 and Lc3 and synthesis of LC3 protein were significantly reduced at the blastocyst stage. Although rapamycin treatment did not affect the developmental rate, it decreased the cell number and increased the rate of apoptosis, and the expression of Atg5, Beclin1 and Lc3 and LC3 protein synthesis were increased. Finally, blastocysts derived following treatment with 3-MA or rapamycin exhibited significantly decreased expression of selected transcription factors, including Pou5f1, Sox2 and Nanog. In conclusion, our results demonstrate that autophagy influences maternal mRNA degradation and apoptosis at the blastocyst stage and suggest that autophagy plays an important role in early embryo development in the pig.
Journal of Reproduction and Development 07/2012; · 1.46 Impact Factor
-
Reproduction Fertility and Development 06/2012; · 2.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Signal transducer and activator of transcription-3 (Stat3) plays a central role in interleukin-6 (IL-6)-mediated cell proliferation by inhibiting apoptosis in a variety of cell types. The Stat3 pathway is essential for embryonic development. The aim of this study was to determine the effects of recombinant IL-6 on the viability and development of porcine diploid parthenotes cultured in vitro. Four-cell parthenotes, derived in vitro, were cultured to the blastocyst stage, with or without recombinant IL-6. The addition of 10 or 100 ng/ml of recombinant swine IL-6 into PZM3 medium increased the development rate of parthenotes to the blastocyst stage (P<0.05). When supplemented with 10 ng/ml of recombinant swine IL-6, the number of parthenotes at the blastocyst stage increased (P<0.05) and apoptosis decreased (P<0.05). Real-time RT-PCR experiments revealed that the addition of recombinant swine IL-6 decreased the mRNA expression of the pro-apoptotic gene Caspase3 (P<0.01) but increased the expression levels of the anti-apoptotic genes Bcl2l1 and Survivin. IL-6 receptors and Stat3 mRNA expression were upregulated after treatment with 10 ng/ml recombinant swine IL-6. Immunoblots and fluorescence labeling experiments showed that the levels of phosphorylated Stat3 were upregulated. These results suggest that recombinant swine IL-6 prevents apoptosis of porcine parthenotes and enhances porcine embryo viability through the IL-6/Stat3 signaling pathway in vitro.
Journal of Reproduction and Development 04/2012; 58(4):453-60. · 1.46 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To investigate the molecular mechanism of mouse oocyte polarity loss during aging.
Experimental study.
Academic basic research laboratory.
Mice.
Oocytes were collected 16 hours after injection of hCG and cultured in M16 medium for an additional 14 hours with or without caffeine.
Expression and localizations of actin nucleators actin-related protein 2/3 complex, JMY, and WAVE2 were examined by immunofluorescence staining, and their messenger RNA levels were examined by real-time reverse transcription-polymerase chain reaction.
The protein and messenger RNA levels of actin-related protein 2/3 complex, JMY, and WAVE2 were decreased in aged oocytes, but the levels were normal in caffeine-treated aged oocytes.
Our data indicated that the loss of oocyte polarity may be due to the degradation of actin nucleators in aged oocytes.
Fertility and sterility 02/2012; 97(4):984-90. · 3.97 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
PLoS ONE 01/2012; 7(12):e52277. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Summary Interspecies intracytoplasmic sperm injection has been carried out to understand species-specific differences in oocyte environments and sperm components during fertilization. While sperm aster organization during cat fertilization requires a paternally derived centriole, mouse and hamster fertilization occur within the maternal centrosomal components. To address the questions of where sperm aster assembly occurs and whether complete fertilization is achieved in cat oocytes by interspecies sperm, we studied the fertilization processes of cat oocytes following the injection of cat, mouse, or hamster sperm. Male and female pronuclear formations were not different in the cat oocytes at 6 h following cat, mouse or hamster sperm injection. Microtubule asters were seen in all oocytes following intracytoplasmic injection of cat, mouse or hamster sperm. Immunocytochemical staining with a histone H3-m2K9 antibody revealed that mouse sperm chromatin is incorporated normally with cat egg chromatin, and that the cat eggs fertilized with mouse sperm enter metaphase and become normal 2-cell stage embryos. These results suggest that sperm aster formation is maternally dependent, and that fertilization processes and cleavage occur in a non-species specific manner in cat oocytes.
Zygote 07/2011; 20(4):371-8. · 1.17 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: During oocyte meiotic maturation, meiotic spindles form in the central cytoplasm and then migrate to the cortex to extrude a small polar body, forming a highly polarized cell through a process involving actin and actin-related molecules. The mechanisms underlying oocyte polarization are still unclear. The Arp2/3 complex regulates oocyte polarization but it is not known whether the WASP family of proteins, a known regulator of the Arp2/3 complex, is involved in this context. In the present study, the role of WASP family member WAVE2 in mouse oocyte asymmetric division was investigated. (1) WAVE2 mRNA and protein were detected during mouse oocyte meiosis. (2) siRNA-mediated and antibody-mediated disruption of WAVE2 resulted in the failure of chromosome congression, spindle formation, spindle positioning and polar body extrusion. (3) WAVE2 regulated actin-driven chromosome migration since chromosomes were arrested in the central cytoplasm by WAVE2 RNAi in the absence of microtubules. (4) Localization of γ-tubulin and MAPK was disrupted after RNAi, confirming the effect of WAVE2 on spindle formation. (5) Actin cap and cortical granule-free domain (CGFD) formation was also disrupted, further confirming the failure of oocyte polarization. Our data suggest that WAVE2 regulates oocyte polarization by regulating meiotic spindle, peripheral positioning, probably via an actin-mediated pathway, and is involved in polar body emission during mouse oocyte meiotic maturation.
Cell cycle (Georgetown, Tex.) 06/2011; 10(11):1853-60. · 5.36 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Low efficiency of somatic cell nuclear transfer (SCNT) is attributed to incomplete reprogramming of transferred nuclei into oocytes. Trichostatin A (TSA), a histone deacetylase inhibitor, has been used to enhance nuclear reprogramming following SCNT. However, the molecular mechanism of TSA for the improvement of the preimplantation embryo and fetal development following SCNT is not known. The present study investigates embryo viability and gene expression of cloned bovine preimplantation embryos in the presence and absence of TSA compared to embryos produced by in vitro fertilization or parthenogenetic activation. Our results indicated that TSA treatment significantly improved total and inner cell mass (ICM) cell number and ratio of ICM:trophectoderm (TE) and also decreased the apoptotic index including total, ICM, and ratio of ICM:TE. Four apoptotic-related genes, Bcl-xL, survivin, Bcl2-associated X protein (Bax), and caspase 3 (Casp3), and four pluripotency/differentiation related genes, Oct4, SRY (sex determining region Y)-box 2 (Sox2), Cdx2, and colony-stimulating factor 1 receptor (Csf1r), were measured by real-time RT-PCR. TSA treatment resulted in the high expression of antiapoptotic gene Bcl-xL and low expression of pro-apoptotic gene Bax compared to untreated NT embryos, fertilized embryos, or parthenotes. Furthermore, mRNA expression of Cdx2 was higher in NT-TSA embryos than in NT and in vitro fertilization (IVF) counterparts. Additionally, low expression of microRNA (mir)-21 in NT embryos was enhanced following TSA treatment. These results suggest that TSA positively regulates nuclear reprogramming, and TSA may increased resistance or reduced signal for induction of apoptosis.
Cellular reprogramming. 04/2011; 13(2):179-89.
-
[show abstract]
[hide abstract]
ABSTRACT: The use of intracytoplasmic sperm injection (ICSI) in model animals is a powerful approach for the study of species-specific fertilization processes and multiploidy embryogenesis. In this study, we examined the fertilization process in mouse oocytes following injection of a single mouse or cat sperm, two mouse spermatozoa or mouse and cat spermatozoa. These treatments did not affect histone H3K9 acetylation or methylation, although the pattern of DNA methylation differed following the injection of cat sperm. Immunocytochemical staining revealed that sperm chromatin was normally incorporated with female mouse chromatin following any of the four injection scenarios. Furthermore, metaphase was successfully entered to reach a normal two-cell stage, and cell division could even persist to produce blastocyst stage embryos. In addition, both mouse and cat Pou5l and Nanog mRNA were expressed in the hybrid embryos. These results suggest that, although epigenetic modification of DNA is affected by the sperm injection treatment, fertilization and cleavage occur in a non-species-specific manner. In addition, despite abnormal division of the chromosomes, intra- and inter-species ICSI produced embryos that could develop into blastocysts.
Journal of Experimental Zoology Part A Ecological Genetics and Physiology 03/2011; 315(6):349-57. · 1.64 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Mitogen-activated protein kinase (MAPK) and maturation/M phase promoting factor (MPF) play crucial roles in oocyte meiotic maturation in mammals. However, the underlying molecular mechanisms have not been addressed. In this study, the effects of the MEK/MAPK pathway inhibitor U0126 and the MPF inhibitor roscovitine on meiotic maturation and maternal gene expression in pig cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) were investigated. Both inhibitors can reversibly block the resumption of meiosis in pig oocytes. COCs or DOs initially cultured in drug-free medium for 24 h and then transferred to medium containing U0126 showed normal progress to the Metaphase II stage (MII); (90.38 vs. 92.16% control group). In contrast, roscovitine treatment from 24 to 44 h significantly inhibited maturation of COCs and DOs. To explore the underlying molecular mechanisms, expression patterns and polyadenylation states of five representative maternal transcripts, cyclin B1, Cdc2, C-mos, GDF9 and BMP15, were examined by real-time PCR and poly(A)-test PCR (PAT assay). U0126 treatment resulted in aberrant expression of Cdc2 and GDF9, while roscovitine significantly maintained all five maternal transcripts at very high levels in treated COCs compared with the control group. The polyadenylation of these mRNAs increased as well. Furthermore, the experiments were repeated in DOs, and the results also indicated that both Cdc2 and GDF9 showed significantly higher expression in both mRNA and polyadenylation levels in the drug treatment groups. Together, these results provide the first demonstration in a mammalian system that MAPK and MPF play important roles in regulation of maternal gene expression during oocyte maturation.
Journal of Reproduction and Development 02/2011; 57(1):49-56. · 1.46 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Mammalian oocyte meiotic maturation involves oocyte polarization and a unique asymmetric division, but until now, the underlying mechanisms have been poorly understood. Arp2/3 complex has been shown to regulate actin nucleation and is widely involved in a diverse range of processes such as cell locomotion, phagocytosis and the establishment of cell polarity. Whether Arp2/3 complex participates in oocyte polarization and asymmetric division is unknown. The present study investigated the expression and functions of Arp2/3 complex during mouse oocyte meiotic maturation. Immunofluorescent staining showed that the Arp2/3 complex was restricted to the cortex, with a thickened cap above the meiotic apparatus, and that this localization pattern was depended on actin. Disruption of Arp2/3 complex by a newly-found specific inhibitor CK666, as well as by Arpc2 and Arpc3 RNAi, resulted in a range of effects. These included the failure of asymmetric division, spindle migration, and the formation and completion of oocyte cytokinesis. The formation of the actin cap and cortical granule-free domain (CGFD) was also disrupted, which further confirmed the disruption of spindle migration. Our data suggest that the Arp2/3 complex probably regulates oocyte polarization through its effect on spindle migration, asymmetric division and cytokinesis during mouse oocyte meiotic maturation.
PLoS ONE 01/2011; 6(4):e18392. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of preimplantation embryos. ESCs exhibit true pluripotency, i.e., the ability to differentiate into cells of all three germ layers in the developing embryo. We used 2-DE MALDI-TOF/TOF to identify differentially expressed proteins among three types of mouse embryonic stem cells (ESCs) derived from fer-tilized, parthenogenetic, and androgenetic (fESC, pESC and aESC, respectively) blastocysts. We detected more than 800 proteins on silver-stained gels of whole protein extracts from each type of ESC. Of these, 52 differentially expressed proteins were identified by the MALDI–TOF/TOF analyzer, including 32 (fESCs vs. pESCs), 28 (fESCs vs. aESCs) and 17 (pESCs vs. aESCs) prominent proteins with significantly higher or lower expression relative to the comparison cells. Among the 32 proteins from fESCs, 12 were significantly increased in expression and 20 were reduced in expression in fESCs com-pared with pESCs. Similarly, 10 of 28 and 8 of 17 proteins were more highly expressed in fESCs and pESCs compared with aESCs, respectively. In contrast, 18 of 28 and 9 of 17 proteins were reduced in expression in fESCs and pESCs compared with aESCs, respectively. Of the eight protein candidates in fESCs, four were in-creased and four were decreased in expression relative to both pESCs and aESCs in the 2-DE analysis. Differential expression of these pro-teins were confirmed by mRNA expression analysis using real time RT-PCR. For three pro-teins, ANXA5, CLIC1 and SRM, Western blot analysis corroborated the expression patterns indicated by the 2-DE results. ANXA5 and CLIC1 were increased in expression and SRM was de-creased in expression in fESCs compared with both pESCs and aESCs. The differentially ex-pressed proteins identified in the present study warrant further investigation towards the goal of their potential application in ESC therapy.
01/2011; 1:1-15.
-
[show abstract]
[hide abstract]
ABSTRACT: Mitochondria are important regulators of both apoptosis and autophagy. One of the triggers for mitochondrial-mediated apoptosis is the production of reactive oxygen species (ROS), which include hydrogen peroxide, superoxide, hydroxyl radical, nitric oxide and peroxynitrite. Recently, several studies have indicated that ROS may also be involved in the induction of autophagy. In the present study, we used H(2)O(2) to induce mitochondrial stress, examined apoptotic- and autophagic-related gene expression and observed LC3 protein (autophagosome presence marker) expression in porcine parthenotes developing in vitro. In porcine four-cell parthenotes cultured for 5 days in NCSU37 medium containing 0.4% BSA, the developmental rate and mitochondrial distribution did not differ from that of the group supplemented with 100 µM H(2)O(2) but was significantly decreased in the group supplemented with 500 µM H(2)O(2) (P<0.05). Transmission electron microscopy (TEM) indicated that whereas normal shaped mitochondria were observed in blastocysts from the control group, abnormal mitochondria (mitophagy) and autophagic vacuoles were observed in blastocysts from the group that received 500 µM H(2)O(2). Furthermore, addition of H(2)O(2) (100 µM and 500 µM) decreased cell numbers (P<0.05) and increased both apoptosis (P<0.05) and LC3 protein expression in the blastocysts. Real-time RT-PCR showed that H(2)O(2) significantly decreased mRNA expression of anti-apoptotic gene Bcl-xL but increased pro-apoptotic genes, Caspase 3 (Casp3) and Bak, and autophagy-related genes, microtubule-associated protein 1 light chain 3 (Map1lc3b) and lysosomal-associated membrane protein 2 (Lamp2). However, the addition of H(2)O(2) had no effect on mRNA expression levels in nuclear DNA-encoded mitochondrial-related genes, cytochrome oxidase (Cox) 5a, Cox5b and Cox6b1, in blastocysts. These results suggest that H(2)O(2) leads to mitochondrial dysfunction that results in apoptosis and autophagy, which is possibly related to porcine early embryo development.
Journal of Reproduction and Development 11/2010; 57(1):143-50. · 1.46 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To evaluate DNA synthesis and epigenetic modification in mouse oocytes during the first cell cycle following the injection of human or hamster sperm.
Mouse oocytes following the injection of human and hamster sperm and cultured in M16 medium.
Male and female pronucleus formation, DNA synthesis, histone protein modification, and heterochromatin formation were similar in mouse oocytes injected with human or hamster sperm. However, DNA methylation patterns were altered in mouse oocytes following human sperm injection. Immunocytochemical staining with a histone H3-MeK9 antibody revealed that human and hamster sperm chromatin associated normally with female mouse chromatin, then entered into the metaphase and formed normal, two-cell stage embryos.
Although differences in epigenetic modification of DNA were observed, fertilization and cleavage occurred in a species non-specific manner in mouse oocytes.
Journal of Assisted Reproduction and Genetics 11/2010; 28(4):325-33. · 1.84 Impact Factor
