Gene expression profiling of porcine skeletal muscle satellite cells after poly(I:C) stimulation.

Porcine satellite cells (PSCs) play a vital role in the construction, development and self-renewal of skeletal muscle. In this study, PSCs were exposed to poly(I:C) stimulation to mimic viral infection during the proliferation and differentiation phases at 0, 12, 24 and 48 hours (h) of the stimulation. The untreated and treated PSCs were analyzed by the RNA-Seq technology. There were 88, 119, 104 and 95 genes being differentially expressed in 0 h vs 12 h treated, 12 h vs 24 h treated, 0 h vs 24 h treated and 24 h vs 48 h untreated comparison libraries, respectively. The GO terms analysis results showed that during the proliferation phase of treated PSCs, the up-regulated genes related to the immune system were highly expressed. In addition, the gene expressions associated with muscle structure development in response to growth factor emerged during the differentiation phase of untreated PSCs. The biological pathways associated with Influenza A, Toll-like receptor and chemokine signaling were revealed in PSCs following poly(I:C) stimulation. The differentially expressed genes were confirmed by quantitative real-time PCR. These findings expanded our understanding of gene expressions and signaling pathways about the infiltrated mechanism of the virus into PSCs.

Ndc80 regulates meiotic spindle organization, chromosome alignment, and cell cycle progression in mouse oocytes.

Ndc80 (called Hec1 in human), the core component of the Ndc80 complex, is involved in regulation of both kinetochore-microtubule interactions and the spindle assembly checkpoint in mitosis; however, its role in meiosis remains unclear. Here, we report Ndc80 expression, localization, and possible functions in mouse oocyte meiosis. Ndc80 mRNA levels gradually increased during meiosis. Immunofluorescent staining showed that Ndc80 was restricted to the germinal vesicle and associated with spindle microtubules from the Pro-MI to MII stages. Ndc80 was localized on microtubules and asters in the cytoplasm after taxol treatment, while Ndc80 staining was diffuse after disruption of microtubules by nocodazole treatment, confirming its microtubule localization. Disruption of Ndc80 function by either siRNA injection or antibody injection resulted in severe chromosome misalignment, spindle disruption, and precocious polar body extrusion. Our data show a unique localization pattern of Ndc80 in mouse oocytes and suggest that Ndc80 may be required for chromosome alignment and spindle organization, and may regulate spindle checkpoint activity during mouse oocyte meiosis.

Regulation of maternal gene expression by MEK/MAPK and MPF signaling in porcine oocytes during in vitro meiotic maturation.

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.

Borealin regulates bipolar spindle formation but may not act as chromosomal passenger during mouse oocyte meiosis.

In mitosis, Borealin is a member of the chromosomal passenger complex (CPC), which plays interaction roles with INCENP and survivin in the complex. Its roles in mammalian meiosis are unknown. Here, we report the expression, localization, and function of Borealin and its relation with survivin in mouse oocyte meiosis. Borealin expression was gradually increased from GV stage to MII. Immunofluorescence results revealed that Borealin accumulated near chromosomes after GVBD, localized at the spindle poles in MI, AI and MII, and at the midbody in TI stage. Taxol and nocodazole treatment showed that the localization of Borealin was dependent on microtubule dynamics, whereas survivin was independent of this. Disruption of Borealin function by antibody injection resulted in severe spindle assembly defects, but did not affect PBE. We also found that depletion of survivin by MO injection had no effect on the localization of Borealin. In conclusion, our data suggest that Borealin is required for bipolar spindle formation, but may not regulate spindle checkpoint activity as a component of the CPC during mouse oocyte meiosis.

Involvement of ER-calreticulin-Ca2+ signaling in the regulation of porcine oocyte meiotic maturation and maternal gene expression.

Calcium is one of the most ubiquitous signaling molecules, and controls a wide variety of cellular processes. It is mainly stored in the endoplasmic reticulum (ER), bound to lumenal proteins. Calreticulin is the major Ca(2+)-binding chaperone in oocytes, and is integral to numerous cellular functions. To better understand the role of the ER- calreticulin-Ca(2+) pathway in oocyte maturation and early embryogenesis, we characterized the porcine calreticulin gene and investigated its expression profile during oocyte maturation and early embryonic development. Calreticulin was widely expressed in pig tissues and its transcripts were downregulated during maturation, especially at 44 hr, and were undetectable at the blastocyst stage. We also investigated the effect of increased cytosolic Ca(2+) induced by the Ca(2+)-ATPase inhibitor, cyclopiazonic acid (CPA), on pig oocyte maturation and maternal gene expression. CPA at 10 microM did not inhibit germinal vesicle breakdown, but did result in the arrest of 38.6% oocytes at or before the MI stage. In addition, expression of the maternal genes C-mos, BMP15, GDF9, and Cyclin B1 was significantly increased in CPA-treated MII oocytes compared with control groups. These data were supported by the results of poly(A)-test PCR, which revealed that the cyclin B1 short isoform (CB-S), GDF9, and C-mos underwent more intensive polyadenylation modification in CPA-treated oocytes than control oocytes, suggesting that polyadenylation may influence Ca(2+)-modulated changes in gene expression. Furthermore, CPA treatment decreased the percentage of four-cell parthenotes that developed into blastocysts, suggesting the need for functional SR/ER Ca(2+)-ATPase pumps or Ca(2+) signals during early embryo development after zygotic genome activation. Together, these data indicate that ER-calreticulin-associated Ca(2+) homeostasis plays a role in oocyte and embryo development, and that alterations in maternal gene expression may contribute to the underlying molecular mechanism, at least partially, via polyadenylation.

Molecular characterization and polyadenylation-regulated expression of cyclin B1 and Cdc2 in porcine oocytes and early parthenotes.

Meiotic maturation of mammalian oocytes is controlled by the maturation/M-phase promotion factor (MPF), a complex of Cdc2 kinase and cyclin B protein. To better understand the molecular mechanism of oocyte maturation, we characterized porcine cyclin B1 and Cdc2 genes, both of which are widely expressed in pig tissues. We further analyzed their expression profiles during in vitro maturation of pig oocyte and early embryonic development at both the mRNA and protein level. Two isoforms of cyclin B1, comprising the same open reading frame but differing in 3'-UTR length, were identified. Cyclin B1 transcripts was up-regulated after 30 hr of maturation, while Cdc2 mRNA levels were unchanged during maturation except for a sharp decline at 44 hr. Cyclin B1 protein synthesis increased with oocyte maturation. Cdc2 protein expression was relatively low during 0-18 hr, followed by a higher level of expression up to 44 hr of maturation. Poly(A)-test PCR clearly revealed that both cyclin B1 isoforms underwent cytoplasmic polyadenylation starting around 18-24 hr during maturation, while a substantial de-adenylation and degradation of Cdc2 isoforms were observed in metaphase II oocytes and during embryo development after parthenogenetic activation. Porcine MII oocytes derived from small follicles (< or = 3 mm) and bad quality 2-cell parthenotes showed lower developmental competence and lower levels of cyclin B1 protein, and Cdc2 mRNA or both gene mRNAs, respectively, compared to their control counterparts. These results suggested that cyclin B1 was regulated posttranscriptionally by cytoplasmic polyadenylation during porcine oocyte maturation. Further, the decreased expression of maternal cyclin B1 and Cdc2 at the mRNA or protein level in developmentally incompetent oocytes and embryos was responsible for, at least in part, a profound defect in further embryonic development.

Involvement of polyadenylation status on maternal gene expression during in vitro maturation of porcine oocytes.

During mammalian oocyte maturation, protein synthesis is mainly controlled through cytoplasmic polyadenylation of stored maternal mRNAs. In this study, the role of polyadenylation modification of maternal transcripts in pig oocytes was investigated by adding cordycepin (3'-dA), a potent polyadenylation inhibitor, to the culture medium of porcine oocytes maturing in vitro. 3'-dA significantly prevented cumulus expansion regardless of the concentration used, and inhibited pig oocyte maturation in a dose-dependent manner. Further, 3'-dA 1 microg/ml-treated MII oocytes experienced significantly lower rates of cleavage (29%) and blastocyst formation (15.35%) compared to control MII oocytes (58.6% and 35.3%, respectively). Western blotting revealed that the activity of mitogen-activated protein kinase (MAPK) and p34(cdc2) was significantly decreased in oocytes and cumulus cells treated with 3'-dA at a concentration of 1 microg/ml or greater. To further explore the underlying molecular mechanisms, expression patterns and polyadenylation states of four important genes, C-mos, cyclin B, GDF9 and BMP15, were studied as representative maternal transcripts by real-time PCR and the PAT assay. 3'-dA at concentrations above 1 microg/ml significantly prevented polyadenylation and caused aberrant expression of C-mos and GDF9 during oocyte maturation. These results suggest that polyadenylation inhibitor blocked pig oocyte maturation in vitro by one or more of the following actions: (1) inactivation of MAPK and MPF in oocytes, especially at the late stages (MI and MII); (2) prevention of cumulus cell expansion through inactivation of cellular MAPK; and (3) inhibition of the maternal mRNA polyadenylation process, which in reverse, disrupted the maternal mRNA patterns in pig oocytes' maturation in vitro.

A link between the interleukin-6/Stat3 anti-apoptotic pathway and microRNA-21 in preimplantation mouse embryos.

Signal transducers and activators 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. MicroRNA-21 (miRNA-21), a ubiquitous miRNA, acts as an anti-apoptotic factor that seems to be indirectly but strictly linked to Stat3. In order to determine whether the IL-6 induced Stat3 anti-apoptosis pathway is linked with miRNA-21, we first determined the effects of recombinant mouse IL-6 on Stat3 expression, mouse embryo viability, and the mRNA levels of apoptosis related genes and miRNA-21 during mouse embryo development in vitro. Addition of 10 or 100 ng/ml of recombinant IL-6 to the culture medium did not affect the developmental ability of 2-cell stage embryos into blastocysts. However, total cell number was significantly increased and apoptosis was reduced in blastocyst stage embryos cultured in the presence of 100 ng/ml of recombinant IL-6. Furthermore, addition of recombinant IL-6 to the culture medium significantly increased the copy numbers of anti-apoptotic miRNA-21, up-regulated Bcl2l1, and down-regulated casp3. Similarly, the injection of mature miRNA-21 into cells up-regulated Bcl2l1 and down-regulated casp3. These results suggest that the induction of the Stat3 anti-apoptotic pathway by IL-6 is linked to miRNA-21 expression, which possibly results in the regulation of cell apoptosis in early mouse embryo development.

Aberrant epigenetic reprogramming of imprinted microRNA-127 and Rtl1 in cloned mouse embryos.

The microRNA (miRNA) genes mir-127 and mir-136 are located near two CpG islands in the imprinted mouse retrotransposon-like gene Rtl1, a key gene involved in placenta formation. These miRNAs appear to be involved in regulating the imprinting of Rtl1. To obtain insights into the epigenetic reprogramming of cloned embryos, we compared the expression levels of mir-127 and mir-136 in fertilized mouse embryos, parthenotes, androgenotes and cloned embryos developing in vitro. We also examined the DNA methylation status of the promoter regions of Rtl1 and mir-127 in these embryos. Our data showed that mir-127 and mir-136 were highly expressed in parthenotes, but rarely expressed in androgenotes. Interestingly, the expression levels of mir-127 and mir-136 in parthenotes were almost twice that seen in the fertilized embryos, but were much lower in the cloned embryos. The Rtl1 promoter region was hyper-methylated in blastocyst stage parthenotes (75.0%), moderately methylated (32.4%) in the fertilized embryos and methylated to a much lower extent (approximately 10%) in the cloned embryos. Conversely, the promoter region of mir-127 was hypo-methylated in parthenogenetically activated embryos (0.4%), moderately methylated (30.0%) in fertilized embryos and heavily methylated in cloned blastocysts (63-70%). These data support a role for mir-127 and mir-136 in the epigenetic reprogramming of the Rtl1 imprinting process. Analysis of the aberrant epigenetic reprogramming of mir-127 and Rtl1 in cloned embryos may help to explain the nuclear reprogramming procedures that occur in donor cells following somatic cell nuclear transfer (SCNT).

[Experimental strategies of the application of RNAi technique in mammalian cells].

As a novel and effective tool for gene function analysis, RNA interference (RNAi) technique has been developed rapidly and made great progress in mammalian cell studies. The mechanisms and experimental operation of RNAi in mammalian cells have become research hotspots, and RNAi mechanisms were supposed to be different from those in plant. In this paper, a review of RNAi mechanisms, the latest research progress and the experimental strategies of RNAi in mammalian cells were presented, including siRNA sequence design, siRNA delivery approaches and RNAi effect detection, so as to offer valuable references for RNAi studies.