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Zhang, Ding-Xiao,Cui, Xiang-Shun,Kim, Nam-Hyung Wiley Subscription Services, Inc., A Wiley Company 2009 Molecular reproduction and development Vol.76 No.9
<P>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 µg/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<SUP>cdc2</SUP> was significantly decreased in oocytes and cumulus cells treated with 3′-dA at a concentration of 1 µg/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 µg/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. Mol. Reprod. Dev. 76: 881–889, 2009. © 2009 Wiley-Liss, Inc.</P>
Zhang, Ding-Xiao,Cui, Xiang-Shun,Kim, Nam-Hyung Wiley Subscription Services, Inc., A Wiley Company 2010 Molecular reproduction and development Vol.77 No.1
<P>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 (≤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. Mol. Reprod. Dev. 77: 38–50, 2010. © 2009 Wiley-Liss, Inc.</P>
Zhang, Ding-Xiao,Li, Xiao-Ping,Sun, Shao-Chen,Shen, Xing-Hui,Cui, Xiang-Shun,Kim, Nam-Hyung Wiley Subscription Services, Inc., A Wiley Company 2010 Molecular reproduction and development Vol.77 No.5
<P>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<SUP>2+</SUP>-binding chaperone in oocytes, and is integral to numerous cellular functions. To better understand the role of the ER– calreticulin–Ca<SUP>2+</SUP> 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<SUP>2+</SUP> induced by the Ca<SUP>2+</SUP>-ATPase inhibitor, cyclopiazonic acid (CPA), on pig oocyte maturation and maternal gene expression. CPA at 10 µM 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<SUP>2+</SUP>-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<SUP>2+</SUP>-ATPase pumps or Ca<SUP>2+</SUP> signals during early embryo development after zygotic genome activation. Together, these data indicate that ER–calreticulin-associated Ca<SUP>2+</SUP> 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. Mol. Reprod. Dev. 77: 462–471, 2010. © 2010 Wiley-Liss, Inc.</P>
Yueyun Ding,Shujiao Zhu,Chaodong Wu,Li Qian,DengTao Li,Li Wang,Yuanlang Wang,Wei Zhang,Min Yang,Jian Ding,Xudong Wu,Xiao-Dong Zhang,Yafei Gao,Zongjun Yin 아세아·태평양축산학회 2019 Animal Bioscience Vol.32 No.7
Objective: Mutations in low-density lipoprotein receptor (LDLR), which encodes a critical protein for cholesterol homeostasis and lipid metabolism in mammals, are involved in cardiometabolic diseases, such as familial hypercholesterolemia in pigs. Whereas microRNAs (miRNAs) can control LDLR regulation, their involvement in circulating cholesterol and lipid levels with respect to cardiometabolic diseases in pigs is unclear. We aimed to identify and analyze LDLR as a potential target gene of SSC-miR-20a. Methods: Bioinformatic analysis predicted that porcine LDLR is a target of SSC-miR-20a. Wild-type and mutant LDLR 3′-untranslated region (UTR) fragments were generated by polymerase chain reaction (PCR) and cloned into the pGL3-Control vector to construct pGL3 Control LDLR wild-3′-UTR and pGL3 Control LDLR mutant-3′-UTR recombinant plasmids, respectively. An miR-20a expression plasmid was constructed by inserting the porcine pre-miR-20a-coding sequence between the HindIII and BamHI sites in pMR-mCherry, and constructs were confirmed by sequencing. HEK293T cells were co-transfected with the miR-20a expression or pMR-mCherry control plasmids and constructs harboring the corresponding 3′-UTR, and relative luciferase activity was determined. The relative expression levels of miR-20a and LDLR mRNA and their correlation in terms of expression levels in porcine liver tissue were analyzed using reverse-transcription quantitative PCR. Results: Gel electrophoresis and sequencing showed that target gene fragments were successfully cloned, and the three recombinant vectors were successfully constructed. Compared to pMR-mCherry, the miR-20a expression vector significantly inhibited wild-type LDLR-3′-UTR-driven (p<0.01), but not mutant LDLR-3′-UTR-driven (p>0.05), luciferase reporter activity. Further, miR-20a and LDLR were expressed at relatively high levels in porcine liver tissues. Pearson correlation analysis revealed that porcine liver miR-20a and LDLR levels were significantly negatively correlated (r = –0.656, p<0.05). Conclusion: LDLR is a potential target of miR-20a, which might directly bind the LDLR 3′-UTR to post-transcriptionally inhibit expression. These results have implications in understanding the pathogenesis and progression of porcine cardiovascular diseases.