Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Cátedra de Química Biológica, Buenos Aires, Argentina; Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Instituto de Investigación y Tecnología en Reproducción Animal (INITRA), Buenos Aires, Argentina.
Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Cátedra de Química Biológica, Buenos Aires, Argentina; Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Instituto de Investigación y Tecnología en Reproducción Animal (INITRA), Buenos Aires, Argentina.
Theriogenology. 2020 May;148:174-179. doi: 10.1016/j.theriogenology.2020.02.033. Epub 2020 Feb 29.
Heparin is the most commonly used in vitro capacitation inducer in the bovine. However, hyaluronic acid (HA) has been recently used for capacitation induction as well as for other reproductive biotechnologies, such as sperm selection and in vitro fertilization (IVF). Our aim was to induce sperm capacitation with heparin or HA in order to study mAC and TK intracellular signals and their relation with cleavage and blastocyst rates after IVF as well as with the oxidative status of the potential bovine embryos. 2,5-dideoxyadenosine and genistein were used as mAC and TK inhibitors, respectively. Sperm capacitation was analyzed using CTC technique, sperm plasma membrane and acrosome integrity were determined using trypan blue stain and differential interference contrast, and mitochondrial activity was evaluated using fluorochrome JC-1. Cleavage rate was analyzed 48h and blastocyst production 7-8 days after IVF, while cytosolic oxidative activity was determined using RedoxSensor Red CC-1 fluorochrome 7h after IVF. When mAC and TK inhibitors were added to sperm samples, only capacitation decreased significantly both in HA and heparin treated samples (P < 0.05), but plasma membrane and acrosome integrity percentages were not affected in any of these groups (P > 0.05). Sperm mitochondrial membrane potential only decreased in heparin treated samples in the presence of both inhibitors (P < 0.05). Oocytes activated with HA sperm treated samples with the addition of 2,5-dideoxyadenosine and genistein presented a lower cytosolic oxidative status than those activated with sperm treated with HA alone (P < 0.05). On the other hand, oocytes activated with heparin treated sperm samples presented a lower cytosolic oxidative status only in the presence of 2,5-dideoxyadenosine (P < 0.05). Therefore, mAC and TK present a differential participation in heparin and HA sperm induced capacitation and mitochondrial function as well as in IVF.
肝素是牛体外最常用的获能诱导剂。然而，透明质酸（HA）最近也被用于获能诱导以及其他生殖生物技术，如精子选择和体外受精（IVF）。我们的目的是用肝素或 HA 诱导精子获能，以研究 mAC 和 TK 细胞内信号及其与 IVF 后卵裂和囊胚率的关系，以及与潜在牛胚胎的氧化状态的关系。2,5-二脱氧腺苷和金雀异黄素分别用作 mAC 和 TK 抑制剂。使用 CTC 技术分析精子获能，使用台盼蓝染色和微分干涉对比法测定精子质膜和顶体完整性，使用荧光染料 JC-1 评估线粒体活性。48 小时后分析卵裂率，IVF 后 7-8 天分析囊胚生成率，而 IVF 后 7 小时使用 RedoxSensor Red CC-1 荧光染料测定胞质氧化活性。当将 mAC 和 TK 抑制剂添加到精子样品中时，只有在 HA 和肝素处理的样品中，获能显著降低（P<0.05），但在这些组中，质膜和顶体完整性百分比均不受影响（P>0.05）。仅当两种抑制剂存在时，肝素处理的样品中的精子线粒体膜电位才会降低（P<0.05）。在添加 2,5-二脱氧腺苷和金雀异黄素的情况下，用 HA 处理的精子处理的卵母细胞的胞质氧化状态低于单独用 HA 处理的卵母细胞（P<0.05）。另一方面，在用肝素处理的精子处理的卵母细胞中，仅在存在 2,5-二脱氧腺苷的情况下，胞质氧化状态才较低（P<0.05）。因此，mAC 和 TK 在肝素和 HA 诱导的精子获能和线粒体功能以及 IVF 中表现出不同的参与。
