Bovine embryo culture in the presence or absence of serum:: Implications for blastocyst development, cryotolerance, and messenger RNA expression
Bovine embryo culture in the presence or absence of serum:: Implications for blastocyst development, cryotolerance, and messenger RNA expression
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DOI：
10.1095/biolreprod.102.007799
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发表时间：
2003-01-01
影响因子：
3.6
通讯作者：
Lonergan, P
中科院分区：
文献类型：
--
作者：
Rizos, D;Gutiérrez-Adán, A;Lonergan, P
我们以前已经表明，虽然卵母细胞的内在质量是影响牛胚胎体外发育过程中囊胚产量的主要因素，但影响囊胚质量的主要因素是受精后培养条件。因此，体外生产的囊胚质量的任何改善都可能来自受精后培养条件的改变。本研究的目的是检查在体外胚胎培养期间存在或不存在血清和BSA浓度对1）卵裂率，2）胚胎发育动力学，3）囊胚产量和4）囊胚质量的影响，通过低温耐受性和基因表达模式进行评估。所有基因转录本的定量通过实时定量逆转录-聚合酶链反应进行。使用来自四个来源的牛囊胚：1）在补充有3 mg/ ml BSA和10%胎牛血清（FCS）的合成输卵管液（SOF）中体外培养，2）在不存在血清的SOF + 3 mg/ml BSA中体外培养，3）在不存在血清的SOF + 16 mg/ml BSA中体外培养，和4）体内囊胚。在第9天，两组间的总体囊胚产量无差异。然而，与3 mg/ml BSA（4.6%，P < 0.001）或16 mg/ml BSA（11.6%，P <0.01）相比，10%血清（20.0%）在第6天时存在显著更多的囊胚。然而，到了第7天，这种差异消失了。玻璃化冷冻后，在存在16 mg/ml BSA或存在10%FCS的情况下产生的囊胚之间的存活率没有差异;在所有时间点和孵化率方面，两组的存活率均显著低于体内对照组。与此相反，在SOF + 3 mg/ml BSA中产生的囊胚在无血清条件下的存活率是中等的，与体内胚胎相比，在72 h时没有差异。在分析的两组囊胚中，发现与凋亡（Bax）、氧化应激（MnSOD、CuZnSOD和SOX）、间隙连接（Cx 31和Cx43）、母体妊娠识别（IFN-tau）以及分化和着床（LIF和LR-β）相关的基因的相对mRNA丰度存在差异。在培养期间血清的存在导致MnSOD、SOX、Bax、LIF和LR-β的表达水平显著增加。Cx 31和Cu/ZnSOD的表达水平也有升高的趋势，但差异不显著。相反，Cx43和IFN-tau的表达水平在血清存在下降低。总之，使用发展能力的测量组合，（卵裂率和囊胚率）和定性测量（如低温耐受性和相对mRNA丰度）来给出修改培养基组成对胚胎的影响的更完整的图片，我们已经表明受精后培养的条件，特别是培养基中血清的存在，会影响胚胎发育的速度和囊胚的质量。在血清存在下产生的胚泡的低温耐受性降低伴随着发育重要基因转录物的相对丰度的偏差。在受精后培养期间省略血清可以显著提高囊胚的低温耐受性，使其达到血清产生的囊胚和体内产生的囊胚之间的中间水平。现在的挑战是努力弥合这一差距。
We have previously shown that, while the intrinsic quality of the oocyte is the main factor affecting blastocyst yield during bovine embryo development in vitro, the main factor affecting the quality of the blastocyst is the postfertilization culture conditions. Therefore, any improvement in the quality of blastocysts produced in vitro is likely to derive from the modification of the postfertilization culture conditions. The objective of this study was to examine the effect of the presence or absence of serum and the concentration of BSA during the period of embryo culture in vitro on 1) cleavage rate, 2) the kinetics of embryo development, 3) blastocyst yield, and 4) blastocyst quality, as assessed by cryotolerance and gene expression patterns. The quantification of all gene transcripts was carried out by real-time quantitative reverse transcription-poly me rase chain reaction. Bovine blastocysts from four sources were used: 1) in vitro culture in synthetic oviduct fluid (SOF) supplemented with 3 mg/ ml BSA and 10% fetal calf serum (FCS), 2) in vitro culture in SOF + 3 mg/ml BSA in the absence of serum, 3) in vitro culture in SOF + 16 mg/ml BSA in the absence of serum, and 4) in vivo blastocysts. There was no difference in overall blastocyst yield at Day 9 between the groups. However, significantly more blastocysts were present by Day 6 in the presence of 10% serum (20.0%) compared with 3 mg/ml BSA (4.6%, P < 0.001) or 16 mg/ml BSA (11.6%, P < 0.01). By Day 7, however, this difference had disappeared. Following vitrification, there was no difference in survival between blastocysts produced in the presence of 16 mg/ml BSA or those produced in the presence of 10% FCS; the survival of both groups was significantly lower than the in vivo controls at all time points and in terms of hatching rate. In contrast, survival of blastocysts produced in SOF + 3 mg/ml BSA in the absence of serum was intermediate, with no difference remaining at 72 h when compared with in vivo embryos. Differences in relative mRNA abundance among the two groups of blastocysts analyzed were found for genes related to apoptosis (Bax), oxidative stress (MnSOD, CuZnSOD, and SOX), communication through gap junctions (Cx31 and Cx43), maternal recognition of pregnancy (IFN-tau), and differentiation and implantation (LIF and LR-beta). The presence of serum during the culture period resulted in a significant increase in the level of expression of MnSOD, SOX, Bax, LIF, and LR-beta. The level of expression of Cx31 and Cu/ZnSOD also tended to be increased, although the difference was not significant. In contrast, the level of expression of Cx43 and IFN-tau was decreased in the presence of serum. In conclusion, using a combination of measures of developmental competence (cleavage and blastocyst rates) and qualitative measures such as cryotolerance and relative mRNA abundance to give a more complete picture of the consequences of modifying medium composition on the embryo, we have shown that conditions of postfertilization culture, in particular, the presence of serum in the medium, can affect the speed of embryo development and the quality of the resulting blastocysts. The reduced cryotolerance of blastocysts generated in the presence of serum is accompanied by deviations in the relative abundance of developmentally important gene transcripts. Omission of serum during the postfertilization culture period can significantly improve the cryotolerance of the blastocysts to a level intermediate between serum-generated blastocysts and those derived in vivo. The challenge now is to try and bridge this gap.