Xing-Hui Shen

Trichostatin A Modulates Apoptotic-Related Gene Expression and Improves Embryo Viability in Cloned Bovine Embryos

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.

Identifying MicroRNA and mRNA Expression Profiles in Embryonic Stem Cells Derived from Parthenogenetic, Androgenetic and Fertilized Blastocysts

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.

Autophagy is required for proper meiosis of porcine oocytes maturing in vitro

Autophagy is an essential cellular mechanism that degrades cytoplasmic proteins and organelles to recycle their components; however, the contribution of autophagy during meiosis has not been studied in porcine oocytes maturing in vitro. In this study, we observed that the autophagy-related gene, LC3, was expressed in porcine oocytes during maturation for 44 h in vitro. Knockdown of the autophagy-related gene, BECN1, reduced both BECN1 and LC3 protein expression levels. Moreover, BECN1 knockdown and treatment with the autophagy inhibitor, LY294002, during maturation of porcine oocytes in vitro impaired polar body extrusion, disturbed mitochondrial function, triggered the DNA damage response, and induced early apoptosis in porcine oocytes. Autophagy inhibition during oocyte maturation also impaired the further developmental potential of porcine oocytes. These results indicate that autophagy is required for the in vitro maturation of porcine oocytes.

265 DICER GENE EXPRESSION IN PRE-IMPLANTATION MOUSE EMBRYOS: IMPLICATION OF TRANSCRIPTION REGULATION AT THE BLASTOCYST STAGE

Dicer is an RNAse III enzyme that is related to the generation of the microRNAs involved in the gene silencing pathway. In order to obtain insight into the role of Dicer in early embryo development, we first evaluated its gene expression levels in mouse oocytes and embryos during in vitro development. The relative abundance of Dicer1 transcripts was established by real-time RT-PCR using the 2-ddCt method. H2a was applied as an internal standard to normalize the real-time RT-PCR reaction efficiency and quantify Dicer1 mRNA. Relatively high expression levels of mRNA in germinal vesicle-stage oocytes steadily decreased up to the 2-cell stage embryo, and then expression remained during morulae and blastocyst formation. Protein synthesis of Dicer was also observed in the mouse oocytes and early embryos. Specific silencing of mRNA expression and protein synthesis by RNA interference (siRNA) did not inhibit developmental events up to the blastocyst (BL) stage. However, Dicer1 siRNA reduced (P <0.05) total nuclei numbers in the BL-stage embryos (Dicer1: 77.2±4.2 vs. control: 62.7±3.1). Real-time RT-PCR also confirmed that, following Dicer1 siRNA microinjection into zygotes, transcription levels of several non-target genes, Cdc42, Cdh1, Dbc2, ILK, Tuba1, Plat, and Tie1, were not changed in blastocyst-stage embryos. However, selected transcription factors, Pou5f1 (P <0.01), Nanog (P <0.005), and Sox2 (P <0.01), in blastocysts were significantly down-regulated. Additionally, POU5F1 protein synthesis was also reduced. Using Applied Biosystem microarray technology, we compared gene expression profiles in control and Dicer1 siRNA microinjected blastocysts. This technique confirmed that 397 or 737 of 16354 genes were up- or down-regulated, respectively, following siRNA microinjection (P <0.05), including 52 transcription factors. The results suggest that expression of Dicer regulates gene expression at the blastocyst-stage embryo for cell fate, possibly by the transcription control.

252 DIFFERENCES IN GENE EXPRESSION PROFILES IN MOUSE FERTILIZED BLASTOCYSTS AND DIPLOID BLASTOCYST STAGE PARTHENOTES

Following parthenogenetic activation, in the absence of a male contribution, oocytes progress into early gestation. To gain insight into the role of the paternal genome during pre-implantation development, we used microarray to compare gene expression profiles in pre-implantation embryos following fertilization and parthenogenetic activation. Fertilized embryos and oocytes were collected from superovulated C57BL/6J female mice. The oocytes were activated with 50 ¼M calcium ionophore A23187 for 5 min. After 5 h of culture in M16 medium with 7.5 ¼g/mL cytochalasin B, oocytes with one polar body and two pronuclei were used in this experiment. The activated oocytes and zygotes were cultured in M16 to the blatocyst stage. Messenger RNA from 50 blastocysts was extracted by means of the Dynabeads mRNA Direct Kit (Dynal, Oslo, Norway), and then linearly amplified for two rounds using the RiboAmp HS RNA Amplification Kit (Arcturus Bioscience, Inc., Mountain View, CA, USA). A set of cRNA targets from the embryos was assembled into a hybridization reaction on the Applied Biosystems 1700 chemiluminescent microarray analyzer (Jung Hwa Scientific Co., Ltd., Seoul, Korea). Each set was repeated three times. All of the correlation coefficients were above 0.9 for experiment replications. Differences in microarray intensities were normalized and grouped by using the Avadis Prophetic 3.3 version, and categories are based on the PANTHER classification system. According to the cDNA microarray data, we additionally categorized genes into transcription- and developmental process-related genes and compared them in both fertilized and parthenogenetically activated blastocysts. Five transcription-related genes (Goosecoid, transcription factor 1, LIM domain, Spi-C transcription factor, and hypoxia inducible factor 3) and seven developmental process related genes (metaxin 1, serine/threonine kinase 22, stromal antigen, butyrophilin, anti-Mullerian hormone type 2 receptor, prolactin-like protein C2, and otoconin 90) were identified in the fertilized blastocysts compared to the blastocyst-stage parthenotes. In contrast, seven transcription- (Amnionless, EHOX-like, calcium signal transducer 2, nuclear receptor 0B, transcription factor CP2, Iroquois related homeobox 3, and zinc finger protein 3) and eight developmental process-related genes (prion protein dublet, X-linked lymphocyte-regulated 3a, muscleblind-like 3, stathmin-like 2, SRY-box-containing gene 7, ephrin B1, muscleblind-like 3, and Iroquois-related homeobox 3) were expressed at a higher level in parthenotes than in fertilized blastocysts. These genes were selected, and their expression levels confirmed, by real-time quantitative RT-PCR. The results indicate that diploid parthenotes at the blastocyst stage may lack or over express genes related to transcription and development processes which possibly result in fetal lethality. Further studies are required to determine whether aberrant gene expression in parthenotes is due to lack of paternal contribution. This work was funded by a grant from the National Research Laboratory Program in Korea.