mrna The Effects of Three Drugs on Maternal mrna Level in Aged Oocytes ZHAO Yun-Cheng 1 LIN Jia-Peng 1 HUANG Jun-Cheng 1 * Chinese Journal of Zoology
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1 Chinese Journal of Zoology ~ mrna * Real-Time PCR Ovis aries mrna Mater Zar1 Dnmt1 GDF9 3 DTT mrna Dnmt1 12 h 3 24 h GDF9 Mater 2 30 h 24 h P < h Zar1 Dnmt1 24 h P > Zar1 DTT 3 P < h P > mrna Zar h P < Mater Zar1 Dnmt1 GDF9 4 mrna DTT DTT mrna S826 A The Effects of Three Drugs on Maternal mrna Level in Aged Oocytes YE Xiao-Fang CHEN Jing-Bo 1 HOU Min 1 WANG Li-Qin 1 ZHAO Yun-Cheng 1 LIN Jia-Peng 1 HUANG Jun-Cheng 1 * 1Key Laboratory of Reproduction & Breeding Biotechnology of Grass Feeding Livestock Ministry of Agriculture Xinjiang Academy of Animal Sciences Urumqi College of Animal Science and Technology Shihezi University Shihezi College of Life Science Xinjiang Normal University Urumqi China Abstract This study used Real-Time PCR technique to quantitatively analyze the expression of the maternal mrna Mater Zar1 Dnmt1 and GDF9 in growth-maturation-aging process of ovine oocytes and investigated the effect of caffeine DTT and vanadate on the level of maternal mrna in aged oocytes respectively. The results showed that the expression of GDF9 Mater and Zar1 decreased gradually from germinal vesicle phase to No No. 2008AA * h_jc@ 163. com YXF001982@ sina. com
2 5 3 4 mrna 155 MⅡ phase while the expression of Dnmt1 decreased gradually from 12 h of maturation culture. The level of all above maternal mrnas was the lowest at MⅡ phase and increased with aging. The expression of Mater Dnmt1 and GDF9 in caffeine treated-or DTT-treated aged oocytes was lower than that of aged oocytes P < but not different from that of matured oocytes P > The expression of Mater Zar1 Dnmt1 and GDF9 in vanadate-treated oocytes was the highest and significantly higher than that of MII group except for Zar1 P < Conclusion the level of Mater Zar1 Dnmt1 and GDF9 is in contrast with oocytes quality. Both caffeine and DTT delay aging progression to some extent and improve the oocyte quality. On the contrary vanadate accelerates aging progression. Key words Caffeine DTT Vanadate Aged oocyte Maternal mrna 5 Dnmtl mrna MⅡ 100 Mus musculus 4 Ovis aries maternal antigen that embryos require Mater 1 zygote arrest 1 Zar1 DNA 1 DNA cytosine-5 methyltransferase 1 Dnmt1 9 growth differentiation factor 9 GDF9 4 Myt1 / Wee1 Mater Mater cdc2 cell division cycle 2 2 MPF Mater cdc25 2 cdc2 MPF Zar1 1 dithiothreitol DTT Zarl 1 3 Zarl Kikuchi 13 maturation promoting factor MPF H1 / p80cdc25 DTT RNA 5 GDF9 p80cdc25 p34cdc2 6 GDF9 MPF DTT 7 GDF9 12 GDF9 8 Dnmtl 30 h DTT Dnmtl
3 156 Chinese Journal of Zoology 45 1 μg + 10 ng / ml EGF mg / ml mmol / L U / ml 1. 3 DTT DTT mmol / L mrna Mater Zar1 Dnmt1 GDF9 18 h 24 h 24 h 30 h 30 h + 24 h + 6 h 24 h 5 mmol / L 30 h DTT DTT + 24 h + 6 h 24 h 1 mmol / L DTT 30 h + 21 h + 6 h 1 21 h 1. 1 TCM- 100 μmol / L 199 Gibco 27 h Sigma Sigma C-0750 DTT Sigma D0632 Wako Pure Chemical Ⅱ Gel Extraction Kit RNA Promega Wizard Plus SV Minipres DNA Purification System DH5α- Pmd-20. T TaKaRa T4 DNA TaKaRa 1. 2 dntps 10 mmol / L 1. 0 μl h PrimeScript Buffer 4. 0 μl Rnase Inhibitor 40 75% U / μl 0. 5 μl Rnase H - 1 μl RNase-free ml dh 2 O 20 μl min min 2 ~ 6 mm min cdna IVM Oligo 6. 0 PCR TCM % FBS v v U / ml FSH + 1 mmol / L 100 μmol / L 18 h 18 h 1. 4 RNA RT-PCR / PBS 3 80 μl 0. 2 ml PCR - 80 Qiagen Reasy Micro 20 μl RNA 0. 5 μg Random 6 mer 25 μmol / L 1. 0 μl min 2 min RNA Microtube Qiagen Reasy Micro RT- PCR QIAEX GenBank 18SrRNA Mater Zar1 Dnmt1 GDF9 1
4 5 3 4 mrna 157 Table S-rRNA Mater Zar1 GDF9 Dnmt1 Primer pairs used in generating ovine 18S-rRNA Mater Zar1 GDF9 and Dnmt1 Genes Primer sequence bp Length Mater Forward 5 -GCTGGAGGCGTGTGGACTG -3 Reverse 5 -GGTCTGTAGATTAGAGGTGGGATGC Zar1 Forward 5 -TGCCGAACATGCCAGAAG-3 Reverse 5 -TCACAGGATAGGCGTTTGC Dnmt1 Forward 5 -CCCCAGGATTACAAGGAAG-3 Reverse 5 -CTGGATGTAACTCGACGTCTCT GDF9 Forward 5 -CCCCAGGATTACAAGGAAG-3 Reverse 5 -CTGGATGTAACTCGACGTCTCT S Forward 5 -GAGAAACGGCTACCACAT C-3 Reverse 5 -GCTATTGGAGCTGGAATT AC PCR 2% QIAEX Ⅱ Gel Extraction Kit pmd20-t PCR OMEGA DNA NCBI 1. 7 Real-time PCR 18S 1 cdna Quantitect SYBY GEEN Qiagen Real-time PCR Quantitative Real-Time PCR 20 μl 2 SYBR premix 10 μl 0. 5 pmol / L cdna 2 μl Rnase-free 7 μl min s s s Light Cycler ng / μl 100 pg / μl 10 pg / μl 1 pg / SPSS mrna μl 100 fg / μl 10 fg / μl 1 fg / μl 7 Mater GDF9 Dnmt1 Zar1 2 Dnmt Tm Mater GDF9 Dnmt1 Zar1 PCR PCR 1 3 NCBI Dnmt1 BLAST Fig. 1 Expression of Mater GDF9 Dnmt1 and Zar1 in ovine oocytes M. 100 bp DNA marker 1. Mater 168 bp 2. GDF9 198 bp 3. Dnmt1 178 bp 4. Zar1 168 bp. Mater GDF9 Dnmt1 Zar1 4 NCBI mrna 100% Quantitative Real-Time PCR
5 158 Chinese Journal of Zoology 45 2 Fig. 2 Dnmt1 Standard curve of Dnmt1 3 Fig. 3 Dnmt1 Melting curve of Dnmt Mater Zar1 Dnmt1 GDF9 4 GDF9 Mater Dnmt1 3 mrna mrna 24 h + 24 h + 6 h P < mrna h 30 h + 24 h + Quantitative Real-Time PCR 6 h 3 Zar1 mrna 4 GDF9 Dnmt1 Mater Zar1 mrna 4 4 P > GDF9 Mater 2. 5 DTT mrna 30 h 24 h P 6 30 h < h P > GDF9 Mater Dnmt1 3 mrna h Zar1 Dnmt1 24 h DTT + 24 h + 6 h P < 24 h P > Zar1 mrna 24 h 30 h h DTT + 24 h + 6 h 3 P > P > h mrna + 24 h + 6 h h DTT + 24 h + 6 h 2. 4 mrna 4 mrna P > 5 30 h 0. 05
6 5 3 4 mrna Fig. 4 GDF9 Dnmt1 Mater Zar1 mrna Relative expression of GDF9 Dnmt1 Mater and Zar1 mrnas in ovine oocytes P < a b c Values with different superscripts indicate significant deviation. Fig. 5 5 mrna Effect of caffeine on level of maternal mrna in aged ovine oocytes 2. 6 mrna 7 24 h 30 h + 21 h + 6 h h + 6 h 4 mrna h + 6 h GDF9 Mater Dnmt1 3 mrna 24 h P 18 - < GDF9 mrna 30 h 19 P < Zar1 mrna 3 mrna P < GDF9 Mater Zar1 Dnmt mrna
7 160 Chinese Journal of Zoology 45 Fig. 6 6 DTT mrna Effect of DTT on level of maternal mrna in aged ovine oocytes Fig. 7 7 mrna Effect of vanadate on level of maternal mrna in aged ovine oocytes 24 h mrna mrna mrna mrna M Ⅱ MPF mrna 15 mrna 3. 2 mrna RNA 20 M Ⅱ MPF 19 /
8 5 3 4 mrna 161 Zar1 4 DTT mrna Real-Time PCR MPF 25 DTT 3 mrna Mater Zar1 Dnmt1 GDF DTT mrna 7 4 mrna Sus scrofa Zar1 3 domestica mrna P < 24 h cyclic adenosine monophosphate camp 30 h 27 h mrna 23 DTT DTT mrna MPF 24 h P < DTT DTT P > DTT MPF 4 Zar1 24 h 30 h DTT 4 Real-Time PCR P > DTT Zar1 Zar1 cdc MPF 27 DTT DTT 2 DTT 3
9 162 Chinese Journal of Zoology 45 DTT MPF MPF mrna MPF Homo sapiens 6 12 Tarin J J Ten J Vendrell F J et al. 28 oocytes. Biol Reprod Tarin J J Perez-Albala S Aguilar A et al. DTT Von Wald T Monisova Y Hacker M R et al. Agerelated variations in follicular apolipoproteins may Tong Z B Gold L Pfeifer K E et al. Mater a maternal influence human oocyte maturation and fertility potential. effect gene required for early embryonic development in Fertil Steril mice. Nat Genet Wu X Viveiros M M Eppig J J et al. Zygote arrest 1 Zar1 is a novel maternal-effect gene critical for the oocyte-to-embryo transition. Nat Genet Wilmut L Schnieke A E McWhir J et al. Viable offspring derived from fetal and adult mammalian cells. Nature Reproduction Update Mater Zar1 19 Miao Y L Kikuchi K Sun Q Y et al. Oocyte aging cellular and molecular changes developmental potential and reversal possibility. Human Reproduction Update -9B / Gilchrist R B Ritter L J Armstrong D T. Oocyte-somatic cell interactions during follicle development in mammals. Anim Reprod Sci / the mouse embryo deficient for dnmt1 distinct effect of hypomethylation on imprinted and random X inactivation. Dev Biol Goud A P Goud P T Diamond M P et al. Nitric oxide delays oocyte aging. Biochemistry Roth Z Aroyo A Yavin S et al. The antioxidant epigallocatechin gallate EGCG moderates the deleterious effects of maternal hyperthermia on follicle-enclosed oocytes in mice. Theriogenology Dithiothreitol prevents age-associated decrease in oocyte / conceptus viability in vitro. Hum Reprod Kikuchi K Naito K Noguchi J et al. Maturation / M- phase promoting factor a regulator of aging in porcine Long-term effects of postovulatory aging of mouse oocytes on offspring a two-generational study. Biol Reprod Navot D Bergh P A Williams M et al. Poor oocyte quality rather tan implantation failure as a cause of agerelated decline in female fertility. Lancet Ginther O J Gasta M O l Gastal E L et al. Effects of age on follicle and hormone dynamics during the oestrous cycle in mares. Reproduction Fertility and Development Tatone C Amicarelli F Carbone M C et al. Cellular and molecular aspects of ovarian follicle ageing. Human Tong Z B Gold L de Pol A. Developmental expression and subcellular localization of mouse Mater an oocytespecific protein essential for early development. Endocrinology Lee J H Campbell K H. Caffeine treatment prevents agerelated changes in ovine oocytes and increases cell numbers in blastocysts produced by somatic cell nuclear transfer. 9 Sado T Fenner M H Tan S S et al. X inactivation in Cloning Stem Cells
10 5 3 4 mrna Maalouf W E Lee J H Campbell K H. Effects of caffeine cumulus cell removal and aging on polyspermy and embryo development on in vitro matured and fertilized ovine oocytes. Theriogenology Kren R Ogushi S Miyano T. Effect of caffeine on meiotic maturation of porcine oocytes. Zygote Mao J Wu G M Prather R S et al. Effect of methylbeta-cyclodextrin treatment of pig spermatozoa on in vitro fertilization and embryo development in the absence or presence of caffeine. Theriogenology Kikuchi K Naito K Noguchi J et al. Maturation / M- phase promoting factor regulates aging of porcine oocytes matured in vitro. Cloning Stem Cells Tarin J J. Potential effects of age-associated oxidative stress on mammalian oocytes / embryos. Mol Hum Reprod Goren S Dekel N. Maintenance of meiotic arrest by a phosphorylated p34cdc2 is independent of cyclic adenosine 3 5 -monophosphate. Biol Reprod Tong Z B Bondy C A Zhou J et al. A human homologue of mouse Mater maternal effect gene essential for early embryonic development. Human Reproduction
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