Cryopreservation damages permeability of sperm mitochondrial membranes, with formation of a mitochondrial permeability transition pore (mPTP). Mitochondria are both a primary synthesis site and principle target for melatonin, which can directly inhibit mPTP formation. The objective was to determine effects of melatonin on mPTP opening of frozen‐thawed ram sperm and elucidate underlying pathways by antagonist and agonists of melatonin receptors (MTs), and antagonists of PI3K and GSK 3β treatments; furthermore, plasma membrane integrity, mitochondrial membrane potential (ΔΨm), mitochondrial cytochrome c (Cyt c) release and fertilization were analysed to assess the effect of mPTP status mediated by melatonin on quality of frozen‐thawed sperm. Fresh ram semen was diluted in glucose‐egg yolk buffer with 0 or 10^–7 M melatonin (frozen and frozen + melatonin groups, respectively) and slow‐frozen. In frozen‐thawed sperm, melatonin added at initiation of 4°C equilibration was most effective for inhibiting mPTP opening, decreasing peptidyl‐prolyl‐cis/trans isomerase activity of cyclophilin D and increasing plasma membrane integrity, ΔΨm, mitochondrial Cyt c concentration and fertilizing ability (p < .05). In a mechanistic study, the melatonin receptor (MT)1 antagonist eliminated inhibition of melatonin on mPTP opening, whereas MT1 agonist had opposite effects (p < .05). Neither MT2 antagonist nor agonist had significant effect, but PI3K and/or GSK 3β antagonist decreased inhibition of MT1 agonist on mPTP opening (p < .05). In conclusion, melatonin improved sperm cryopreservation, perhaps by acting on MT1 via the PI3K‐Akt‐GSK 3β pathway to inhibit mPTP opening.

中文翻译：

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褪黑素通过抑制线粒体通透性过渡孔的开放来改善ram精子的冷冻保存。

冷冻保存会破坏精子线粒体膜的通透性，并形成线粒体通透性过渡孔（mPTP）。线粒体既是褪黑素的主要合成位点，又是主要靶标，可直接抑制mPTP的形成。目的是确定褪黑激素对冷冻融化的精子的mPTP开放的影响，并通过褪黑激素受体（MTs）的拮抗剂和激动剂以及PI3K和GSK3β治疗的拮抗剂阐明其基本途径；此外，还分析了质膜完整性，线粒体膜电位（ΔΨm），线粒体细胞色素c（Cyt c）释放和受精情况，以评估褪黑素介导的mPTP状态对冻融精子质量的影响。将新鲜的公羊精液在葡萄糖-蛋黄缓冲液中稀释为0或10 ^–7 M褪黑激素（分别为冷冻和冷冻+褪黑素组）和慢速冷冻。在冷冻解冻的精子中，在4°C平衡开始时添加的褪黑素最有效地抑制mPTP的开放，降低亲环蛋白D的肽基脯氨酰顺式/反式异构酶活性并增加质膜完整性，ΔΨm，线粒体Cyt c浓度和施肥能力（p <  .05）。在一项机理研究中，褪黑激素受体（MT）1拮抗剂消除了褪黑激素对mPTP开放的抑制作用，而MT1激动剂则具有相反的作用（p  <.05）。MT2拮抗剂和激动剂均无明显作用，但PI3K和/或GSK3β拮抗剂可降低MT1激动剂对mPTP开放的抑制作用（p < .05）。总之，褪黑激素可以改善精子的冷冻保存能力，也许是通过PI3K-Akt-GSK3β途径作用于MT1来抑制mPTP的开放。