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Published on: September 19, 2011
Melatonin improves cryopreservation of ram sperm by inhibiting mitochondrial permeability transition pore opening
Yi Fang1, Chengzhen Zhao1, Hai Xiang1
1Jilin Provincial Key Laboratory of Grassland Farming, Northeast Institute of Geography and Agoecology, Chinese Academy of Sciences, Changchun, Jilin, China.
Abstract:
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.
Insights
Melatonin protects ram sperm during cryopreservation by inhibiting mitochondrial permeability transition pore (mPTP) opening. This improves sperm quality and fertilizing ability, potentially via the MT1 receptor and PI3K-Akt-GSK 3β pathway.
Area of Science:
- Reproductive Biology
- Mitochondrial Physiology
- Cryobiology
Background:
- Cryopreservation induces mitochondrial membrane damage in sperm, leading to the opening of the mitochondrial permeability transition pore (mPTP).
- Melatonin, a hormone, is known to interact with mitochondria and can inhibit mPTP formation.
Purpose of the Study:
- To investigate the protective effects of melatonin on frozen-thawed ram sperm, specifically its impact on mPTP opening.
- To elucidate the molecular pathways involved in melatonin's action using receptor antagonists/agonists and signaling pathway inhibitors.
Main Methods:
- Ram semen was cryopreserved with or without melatonin.
- Frozen-thawed sperm quality was assessed by measuring plasma membrane integrity, mitochondrial membrane potential (ΔΨm), and cytochrome c (Cyt c) release.
- Melatonin receptor (MT1, MT2) antagonists/agonists and PI3K/GSK 3β inhibitors were used to explore the underlying mechanisms.
Main Results:
- Melatonin significantly inhibited mPTP opening and reduced cyclophilin D activity in frozen-thawed ram sperm.
- Melatonin treatment increased plasma membrane integrity, ΔΨm, mitochondrial Cyt c concentration, and fertilizing ability.
- The protective effect of melatonin was mediated by the MT1 receptor and involved the PI3K-Akt-GSK 3β signaling pathway.
Conclusions:
- Melatonin enhances sperm cryopreservation by inhibiting mPTP opening.
- The MT1 receptor and the PI3K-Akt-GSK 3β pathway are crucial for melatonin's protective effects on sperm quality post-thawing.
