Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
Portalagre Polytechnic Institute, Superior Agriculture School of Elvas, Elvas, Portugal.
PLoS One. 2019 Jul 5;14(7):e0211994. doi: 10.1371/journal.pone.0211994. eCollection 2019.
The population of stallion spermatozoa that survive thawing experience compromised mitochondrial functionality and accelerated senescence, among other changes. It is known that stallion spermatozoa show very active oxidative phosphorylation that may accelerate sperm senescence through increased production of reactive oxygen species. Rosiglitazone has been proven to enhance the glycolytic capability of stallion spermatozoa maintained at ambient temperature.
Thus, we hypothesized that thawed sperm may also benefit from rosiglitazone supplementation.
Thawed sperm were washed and resuspended in Tyrodes media, and the samples were divided and supplemented with 0 or 75 μM rosiglitazone. After one and two hours of incubation, mitochondrial functionality, Akt phosphorylation and caspase 3 activity were evaluated. Additional samples were incubated in the presence of an Akt1/2 inhibitor, compound C (an AMPK inhibitor) or GW9662 (an antagonist of the PPARγ receptor).
Rosiglitazone maintained Akt phosphorylation and reduced caspase 3 activation (p<0.01), both of which were prevented by incubation in the presence of the three inhibitors. Rosiglitazone also enhanced mitochondrial functionality (P<0.01).
We provide the first evidence that the functionality of frozen stallion spermatozoa can be potentially improved after thawing through the activation of pro survival pathways, providing new clues for improving current sperm biotechnology.
解冻后存活的种马精子经历了线粒体功能受损和加速衰老等变化。已知种马精子表现出非常活跃的氧化磷酸化，通过增加活性氧的产生可能加速精子衰老。罗格列酮已被证明可以增强在环境温度下保存的种马精子的糖酵解能力。
因此，我们假设解冻后的精子也可能受益于罗格列酮的补充。
解冻的精子被洗涤并重新悬浮在 Tyrodes 培养基中，将样品分为补充 0 或 75 μM 罗格列酮的两组。孵育 1 小时和 2 小时后，评估线粒体功能、Akt 磷酸化和 caspase 3 活性。将其他样品在 Akt1/2 抑制剂化合物 C（AMPK 抑制剂）或 GW9662（PPARγ 受体拮抗剂）存在下孵育。
罗格列酮维持 Akt 磷酸化并减少 caspase 3 活化（p<0.01），这两种作用都被三种抑制剂的存在所阻止。罗格列酮还增强了线粒体功能（P<0.01）。
我们首次提供了证据，表明通过激活生存相关途径，冷冻种马精子的功能在解冻后可能得到改善，为改进当前的精子生物技术提供了新的线索。
