Ríos G L, Buschiazzo J, Mucci N C, Kaiser G G, Cesari A, Alberio R H
Biotecnología de la Reproducción, Departamento de Producción Animal, INTA, Balcarce, Buenos Aires, Argentina.
Biotecnología de la Reproducción, Departamento de Producción Animal, INTA, Balcarce, Buenos Aires, Argentina; Instituto de Investigaciones Bioquímicas de Bahía Blanca, Universidad Nacional del Sur, Consejo Nacional de Investigaciones Científicas y Técnicas (UNS-CONICET), Bahía Blanca, Buenos Aires, Argentina.
Theriogenology. 2015 Mar 15;83(5):874-80. doi: 10.1016/j.theriogenology.2014.11.022. Epub 2014 Nov 26.
The conditions for in vitro oocyte maturation impact on cytoplasmic and nuclear processes in the oocyte. These events are differentially influenced by the nature of the maturation inducer and the presence of intact cumulus in cumulus-oocyte complexes. Epidermal growth factor is the main growth factor promoting oocyte maturation. Also, hyaluronic acid (HA) produced by cumulus cells is known to be responsible for the correct structural and functional organization of the cumulus during oocyte maturation. Therefore, we evaluated the developmental competence of bovine oocytes matured in vitro in a maturation medium supplemented with both EGF and HA, compared to FSH and fetal bovine serum (FBS). In addition, the impact of IVM conditions on the proteomic profile of metaphase II bovine oocytes was analyzed by two-dimensional electrophoresis. Cumulus-oocyte complexes were matured in two media: (1) 10 ng/mL EGF, 15 μg/mL HA, and 100-μM cysteamine and (2) 0.01 UI/mL rh-FSH and 10% FBS. The percentages of first polar body and embryo production and the kinetics of embryo development and oocyte proteomic profiles were analyzed. Oocytes matured in the presence of EGF-HA showed an increase (6%, P < 0.05) in the percentage of polar body extrusion. The blastocyst rate was 3% (P < 0.05) higher in the FSH-FBS group, but no differences were found in the rate of expanded blastocyst neither in total embryo production between IVM conditions. Cleavage rate of oocytes matured with FSH-FBS was 5% higher (P < 0.05) with respect to EGF-HA-matured oocytes when evaluated 30 hours after fertilization. However, at Day 7, those inseminated oocytes that underwent division at a correct timing showed that although there are still early blastocysts in the FSH-FBS condition, EGF-HA embryos have developed completely into blastocysts. Still, the production rate of those embryos that achieved expansion was similar between both maturation conditions. On the other hand, noncleaved presumptive zygotes at Day 7 developed into the different stages with similar rates (∼4%) independently of the medium condition. Modifications of IVM medium composition markedly affected protein profile of bovine oocytes in a differential manner. The proteomic approach revealed the presence of 68 spots in both treatments, 41 exclusively found in the FSH-FBS group and 64 exclusive for the EGF-HA group. Taken together, these results indicate that combined EGF-HA supplementation of in vitro maturation medium could be used to improve oocyte meiotic competence and ensure a better timing to develop into the blastocyst stage.
体外卵母细胞成熟的条件会影响卵母细胞中的细胞质和细胞核过程。这些事件受到成熟诱导剂的性质以及卵丘 - 卵母细胞复合体中完整卵丘的存在的不同影响。表皮生长因子是促进卵母细胞成熟的主要生长因子。此外，已知卵丘细胞产生的透明质酸（HA）负责卵母细胞成熟过程中卵丘的正确结构和功能组织。因此，我们评估了与促卵泡素（FSH）和胎牛血清（FBS）相比，在补充了表皮生长因子（EGF）和透明质酸（HA）的成熟培养基中体外成熟的牛卵母细胞的发育能力。此外，通过二维电泳分析了体外成熟（IVM）条件对中期II期牛卵母细胞蛋白质组图谱的影响。卵丘 - 卵母细胞复合体在两种培养基中成熟：（1）10 ng/mL表皮生长因子、15 μg/mL透明质酸和100 μM半胱胺，以及（2）0.01 UI/mL重组人促卵泡素（rh - FSH）和10%胎牛血清。分析了第一极体和胚胎产生的百分比、胚胎发育动力学以及卵母细胞蛋白质组图谱。在表皮生长因子 - 透明质酸存在下成熟的卵母细胞极体排出百分比增加了6%（P < 0.05）。FSH - FBS组的囊胚率高3%（P < 0.05），但在体外成熟条件之间，扩张囊胚率和总胚胎产量均未发现差异。受精后30小时评估时，用FSH - FBS成熟的卵母细胞的分裂率比用表皮生长因子 - 透明质酸成熟的卵母细胞高5%（P < 0.05）。然而，在第7天，那些在正确时间进行分裂的受精卵母细胞表明，尽管在FSH - FBS条件下仍有早期囊胚，但表皮生长因子 - 透明质酸组的胚胎已完全发育成囊胚。尽管如此，两种成熟条件下实现扩张的胚胎的产生率相似。另一方面，第7天未分裂的假定合子以相似的速率（约4%）发育到不同阶段，与培养基条件无关。体外成熟培养基成分的改变以不同方式显著影响牛卵母细胞的蛋白质图谱。蛋白质组学方法显示两种处理中均存在68个斑点，其中41个仅在FSH - FBS组中发现，64个为表皮生长因子 - 透明质酸组所特有。综上所述，这些结果表明，在体外成熟培养基中联合补充表皮生长因子和透明质酸可用于提高卵母细胞的减数分裂能力，并确保更好地发育到囊胚阶段的时间。
