National Agricultural Research Center for Kyushu Okinawa Region, Kumamoto, Japan.
Mol Reprod Dev. 2010 May;77(5):439-48. doi: 10.1002/mrd.21164.
Recently, the quantity of cathepsin transcripts in cumulus cells was found to be associated with low-developmental competence of bovine oocytes. In the present study, we investigated (1) the relation between cathepsin B activity and the quality of in vitro-matured cumulus-oocyte complexes (IVM COCs) and denuded oocytes and (2) the effect of a cathepsin B inhibitor (E-64) on embryo development and quality. The activity of cathepsin B was evaluated in IVM COCs and denuded oocytes. After maturation of COCs with or without E-64, followed by in vitro fertilization, zygotes were cultured for 8 days. Cleavage and blastocyst rates were evaluated on days 2 and 8, respectively. Quality of embryos was evaluated by differential staining of day 8 blastocysts. TUNEL staining was conducted on IVM COCs and blastocysts. Cathepsin B activity was clearly detected in the low-quality oocytes, and in the cumulus cells of both high- and low-quality oocytes. This latter activity was diminished by addition of E-64. The presence of E-64 during IVM also significantly increased both the blastocyst rate and the total cell number, and improved blastocyst quality associated with a significant increase of trophoectoderm cells. TUNEL staining revealed that inhibition of cathepsin B significantly decreased the number of apoptotic nuclei in both the cumulus cell layer of matured oocytes and blastocysts. These results indicate that cathepsin B activity can be a useful marker of oocyte quality. Furthermore, inhibition of cathepsin B greatly improves the developmental competence of bovine oocytes and increases the number of high-quality embryos.
最近，发现卵丘细胞中的组织蛋白酶转录本数量与牛卵母细胞的低发育能力有关。在本研究中，我们研究了（1）组织蛋白酶 B 活性与体外成熟卵丘-卵母细胞复合物（IVM COCs）和去卵丘卵母细胞质量之间的关系，以及（2）组织蛋白酶 B 抑制剂（E-64）对胚胎发育和质量的影响。评估了 IVM COCs 和去卵丘卵母细胞中的组织蛋白酶 B 活性。在 COCs 成熟过程中加入或不加入 E-64 后，进行体外受精，将受精卵培养 8 天。分别在第 2 天和第 8 天评估卵裂率和囊胚率。通过对第 8 天囊胚的差异染色评估胚胎质量。对 IVM COCs 和囊胚进行 TUNEL 染色。在低质量卵母细胞以及高质量和低质量卵母细胞的卵丘细胞中均清晰检测到组织蛋白酶 B 活性。添加 E-64 可减少这种活性。在 IVM 过程中添加 E-64 还显著提高了囊胚率和总细胞数，并改善了与滋养外胚层细胞显著增加相关的囊胚质量。TUNEL 染色表明，抑制组织蛋白酶 B 显著减少了成熟卵母细胞和囊胚卵丘细胞层中凋亡核的数量。这些结果表明，组织蛋白酶 B 活性可以作为卵母细胞质量的有用标志物。此外，抑制组织蛋白酶 B 可显著提高牛卵母细胞的发育能力，并增加高质量胚胎的数量。
