Characteristics of Preimplantational Development of Porcine Parthenogenetic Diploids Relative to the Existence of Amino Acids In Vitro1
Characteristics of Preimplantational Development of Porcine Parthenogenetic Diploids Relative to the Existence of Amino Acids In Vitro1
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DOI：
10.1095/biolreprod.102.004812
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发表时间：
2002-12
期刊：
影响因子：
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通讯作者：
N. Van Thuan;H. Harayama;M. Miyake
中科院分区：
文献类型：
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作者：
N. Van Thuan;H. Harayama;M. Miyake
摘要本研究旨在研究氨基酸对体外成熟卵母细胞电刺激（El-St）和细胞松弛素B （cytochalasin B）处理产生的猪孤雌二倍体体外发育的影响。发育培养基以Whitten培养基为基础，含有0.5 mg/ml透明质酸（mWM），采用两步培养系统，在4细胞期（El-St后48或72小时）之前使用290 mOsmol，随后使用256 mOsmol直到囊胚期（mWMs）。在试验1中，二倍体在添加0.01 ~ 5 mg/ml聚乙烯醇（PVA）的mWMs中培养168 h。试验2将二倍体分别在含0.5 mg/ml PVA （PVA-mWMs）的培养基中培养0、48和72 h，然后在不含谷氨酰胺（E-AA）的Eagle基础培养基中添加必需氨基酸，在最低必需培养基（NE-AA）中添加非必需氨基酸的PVA-mWMs中培养168 h。结果表明，在添加0.05 ~ 5.0 mg/ml PVA的mWMs中，二倍体可以发育到囊胚期（49% ~ 53% vs. 63%, P < 0.05），但单独用PVA替代BSA不能支持囊胚的扩增（11% ~ 20% vs. 39%, P < 0.05）或囊胚的增殖。在PVA-mWMs中添加E- aa和NE-AA （E+NE-AA）对二倍体发育到囊胚期有明显的抑制作用。然而，在El-St后48或72 h，在PVA-mWMs中添加E+NE-AA可以很好地支持二倍体发育到囊胚期，并支持囊胚的扩增。在实验3-5中，测定了E-AA中哪些氨基酸类型在El-St后48 h内抑制了二倍体的发育。实验6观察E-AA和/或NE-AA在4细胞期后的刺激作用。这些实验结果清楚地表明，非极性E-AA的存在，特别是缬氨酸、亮氨酸、异亮氨酸和蛋氨酸的存在，在El-St后的前48小时内，导致了第一次分裂的严重延迟和4细胞期以后发育的抑制。4细胞期后NE-AA的存在有利于囊胚的扩增（33%），而E-AA的存在增加了压实后二倍体的分裂率，囊胚细胞总数（53.7±2.7）个，内细胞质量（12±0.5）个。这些结果表明，非极性E-AA在无蛋白培养基中存在48 h会导致猪孤雌生殖二倍体的4细胞阻滞。
Abstract The present study was designed to investigate the effects of amino acids on the in vitro development of porcine parthenogenetic diploids that were produced by electrostimulation (El-St) and cytochalasin B treatment of in vitro-matured oocytes. The culture medium for development, based on Whitten medium, contained 0.5 mg/ml of hyaluronic acid (mWM), and a two-step culture system in which 290 mOsmol before the 4-cell stage (48 or 72 h after El-St) and, subsequently, 256 mOsmol up to the blastocyst stage (mWMs) were used. In experiment 1, the diploids were cultured for 168 h in mWMs supplemented with 0.01–5 mg/ml of polyvinyl alcohol (PVA). In experiment 2, the diploids were cultured in mWMs containing 0.5 mg/ml of PVA (PVA-mWMs) for 0, 48, or 72 h and then cultured for 168 h after El-St in PVA-mWMs supplemented with essential amino acids for Eagle basal medium without glutamine (E-AA) and nonessential amino acids for minimum essential medium (NE-AA). The results showed that diploids can develop up to the blastocyst stage in mWMs including 0.05–5.0 mg/ml of PVA (49%–53% vs. 63%, P > 0.05), but the replacement of BSA with PVA alone could not support the expansion of blastocysts (11%–20% vs. 39%, P < 0.05) or their proliferation. The addition of both E-AA and NE-AA (E+NE-AA) to PVA-mWMs from the 1-cell stage resulted in severe inhibition of the development of diploids to the blastocyst stage. However, the addition of E+NE-AA to PVA-mWMs later than 48 or 72 h after El-St well supported the development of diploids to the blastocyst stage and supported the expansion of blastocysts. In experiments 3–5, which types of amino acids in E-AA inhibited the development of diploids during the first 48 h after El-St were determined. In experiment 6, the stimulatory effects of E-AA and/or NE-AA after the 4-cell stage were examined. The results of those experiments clearly showed that the presence of nonpolar E-AA, especially for valine, leucine, isoleucine, and methionine, during the first 48 h after El-St caused severe delay of the first division and inhibition of development beyond the 4-cell stage. The presence of NE-AA after the 4-cell stage produced a favorable condition for the expansion of blastocysts (33%), whereas the presence of E-AA increased the cleavage rates of the diploids after compaction and the total number of cells in the blastocysts (53.7 ± 2.7) and inner cell mass (12 ± 0.5). These findings indicate that the presence of nonpolar E-AA in a protein-free medium during the first 48 h causes the 4-cell block in porcine parthenogenetic diploids.