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在添加生长因子和合成大分子的α-MEM中成熟的卵母细胞来源的牛囊胚中,植入前发育以及热休克蛋白70(Hsp-70)和Bax基因的表达

Preimplantation development and expression of Hsp-70 and Bax genes in bovine blastocysts derived from oocytes matured in alpha-MEM supplemented with growth factors and synthetic macromolecules.

作者信息

Vireque A A, Camargo L S A, Serapião R V, Rosa E Silva A A M, Watanabe Y F, Ferreira E M, Navarro P A A S, Martins W P, Ferriani R A

机构信息

Department of Obstetrics and Gynecology, School of Medicine of Ribeirão Preto, University of São Paulo, Bandeirantes anevue 3900, 14049-900 Ribeirão Preto, SP, Brazil.

出版信息

Theriogenology. 2009 Mar 1;71(4):620-7. doi: 10.1016/j.theriogenology.2008.09.028. Epub 2008 Oct 28.

Abstract

In vitro culture conditions affect both the maternal and embryonic expression of genes and is likely to alter both oocyte and embryo developmental competence. The search for better and less variable culture conditions simulating those in vivo has led to the development of defined culture media, with lower impact on the molecular reprogramming of oocytes and embryos. We evaluated embryo development and relative abundance (RA) of Hsp-70 and Bax transcripts in bovine blastocysts produced from oocytes matured in a chemically defined IVM system with synthetic polymers. Immature cumulus oocyte complexes (COCs) were matured for 22-24h in alpha-MEM supplemented with IGF-1, insulin, 0.1% polyvinyl alcohol (PVA), or 0.1% polyvinylpyrrolidone (PVP), but without FSH or LH. The control group consisted of COCs matured in TCM plus FSH and 10% estrous cow serum. After fertilization, presumptive zygotes were co-cultured with cumulus cells until 224 h post-insemination. Total RNA was isolated from embryo pools, reverse transcribed into cDNA, and subjected to transcript analysis by real-time PCR. Cleavage rate was higher (P<0.05) for the control group (68.3%) than for the PVA (54.4%) and PVP-40 (58.3%) groups. Nevertheless, there was no difference among the PVA, PVP-40 and control groups in blastocyst or hatching rates. Similarly, no difference in relative abundance of Hsp-70 and Bax transcripts was detected in comparison to the control group. We inferred that bovine oocytes can be matured in serum- and gonadotrophin-free medium supplemented with PVA or PVP, enriched with IGF-I and insulin, without altering post-cleavage development and relative abundance of some genes associated with stress and apoptosis.

摘要

体外培养条件会影响基因在母体和胚胎中的表达,并且可能改变卵母细胞和胚胎的发育能力。寻找更好且变异性更小、模拟体内情况的培养条件促使了限定培养基的开发,这种培养基对卵母细胞和胚胎的分子重编程影响较小。我们评估了在含有合成聚合物的化学限定体外成熟(IVM)系统中成熟的卵母细胞所产生的牛囊胚中Hsp - 70和Bax转录本的胚胎发育及相对丰度(RA)。未成熟的卵丘卵母细胞复合体(COCs)在添加了IGF - 1、胰岛素、0.1%聚乙烯醇(PVA)或0.1%聚乙烯吡咯烷酮(PVP)但不含促卵泡素(FSH)或促黄体生成素(LH)的α - MEM中成熟22 - 24小时。对照组由在添加FSH和10%发情母牛血清的TCM中成熟的COCs组成。受精后,假定的合子与卵丘细胞共培养至授精后224小时。从胚胎池中分离总RNA,逆转录成cDNA,并通过实时PCR进行转录本分析。对照组的卵裂率(68.3%)高于PVA组(54.4%)和PVP - 40组(58.3%)(P<0.05)。然而,PVA组、PVP - 40组和对照组在囊胚率或孵化率上没有差异。同样,与对照组相比,未检测到Hsp - 70和Bax转录本相对丰度的差异。我们推断,牛卵母细胞可以在添加了PVA或PVP、富含IGF - I和胰岛素的无血清和无促性腺激素培养基中成熟,而不会改变卵裂后的发育以及一些与应激和凋亡相关基因的相对丰度。

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