Animal Physiology and Reproduction Division, ICAR- Central Institute for Research on Buffaloes, Hisar, 125001, Haryana, India; Department of Veterinary Gynaecology and Obstetrics, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, 125001, Haryana, India.
Animal Physiology and Reproduction Division, ICAR- Central Institute for Research on Buffaloes, Hisar, 125001, Haryana, India.
Theriogenology. 2023 Jul 1;204:31-39. doi: 10.1016/j.theriogenology.2023.04.001. Epub 2023 Apr 6.
It is a known fact that cryopreservation initiates premature capacitation in spermatozoa during the cryopreservation process. Protein tyrosine phosphorylation is a landmark of cascade reaction accountable for capacitation or capacitation-like changes in spermatozoa. Therefore, our hypothesis was to test an inhibitor (H89) that reversibly inhibits the cascade reaction responsible for capacitation during the cryopreservation process but does not hamper normal capacitation and fertilizing ability of sperm. For this, sixteen ejaculates were collected from Murrah buffalo bulls (n = 4). Each ejaculate was divided into four equal aliquots and diluted in an egg yolk-based semen dilutor supplemented with 0, 2, 10, and 30 μM concentrations of H89 and cryopreserved. Interestingly, H89 reduces cholesterol efflux from spermatozoa and protects spermatozoa from membrane damage during the cryopreservation process. H89 did not prevent lipid peroxidation of the sperm membrane. H89 reduced intracellular calcium concentration in spermatozoa in a dose-dependent manner, but tyrosine phosphorylation reduction was observed in the 2 and 10 μM H89 groups. The CTC assay revealed that the percentage of uncapacitated spermatozoa in different treatment groups increases in a dose-dependent manner. In the in vitro capacitation medium, the effect of H89 is abolished and spermatozoa underwent normal capacitation, but H89-treated spermatozoa attached to zona pellucida in large numbers compared to untreated spermatozoa. In conclusion, H89 does not only inhibit tyrosine phosphorylation of spermatozoa but it reduces cholesterol efflux and calcium influx, and ultimately reduces capacitation-like changes during the cryopreservation process.
这是一个已知的事实，即冷冻保存会在冷冻保存过程中引发精子的过早获能。蛋白酪氨酸磷酸化是精子获能或类似获能变化的级联反应的标志。因此，我们的假设是测试一种抑制剂（H89），它可以可逆地抑制冷冻保存过程中负责获能的级联反应，但不影响精子的正常获能和受精能力。为此，从 Murrah 水牛公牛（n=4）中收集了 16 份精液。每份精液被分成四个相等的等分，并在含有 0、2、10 和 30μM H89 的蛋黄基础精液稀释剂中稀释，并进行冷冻保存。有趣的是，H89 减少了精子中的胆固醇外排，并在冷冻保存过程中保护精子免受膜损伤。H89 没有阻止精子膜的脂质过氧化。H89 以剂量依赖的方式降低精子内的钙离子浓度，但在 2 和 10μM H89 组中观察到酪氨酸磷酸化减少。CTC 检测显示，不同处理组的未获能精子百分比呈剂量依赖性增加。在体外获能培养基中，H89 的作用被消除，精子经历了正常的获能，但与未经处理的精子相比，H89 处理的精子大量附着在透明带。总之，H89 不仅抑制精子的酪氨酸磷酸化，还降低胆固醇外排和钙离子内流，最终减少冷冻保存过程中的类似获能变化。
