Melatonin improves cryopreservation of ram sperm by inhibiting mitochondrial permeability transition pore opening

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.

Melatonin improves rate of monospermic fertilization and early embryo development in a bovine IVF system

“…The effects of melatonin on mitochondrial function have been investigated in spermatozoa from various mammalian species, including ram [ 62 , 63 ] and human [ 12 ]. A recent study reported a higher mitochondrial respiratory capacity of sperm cells by increasing ATP production via the oxidative phosphorylation pathway after melatonin treatment [ 63 ].…”

The mitochondrial permeability transition pore: an evolving concept critical for cell life and death

“…Benefits of melatonin are also known in the heart from aged mice and in in frozen-thawed sperm, where melatonin administration inhibited mPTP opening and improved cellular respiration and ATP production (Sahach et al, 2008;Fang et al, 2019). A recent mechanistic study revealed that a melatonin receptor 1 (MT1) antagonist eliminated the mPTP opening inhibition generated by melatonin, whereas a MT1 agonist had the opposite effect (Fang et al, 2020). However, the exact mechanism of action by which melatonin regulates the mPTP still remains unclear, although related molecular mechanisms have been proposed.…”

Melatonin as an Antioxidant and Immunomodulator in Atopic Dermatitis—A New Look on an Old Story: A Review

“…In mitochondria, MT seems to be the main executor of this role [ 60 ]. MT is able to protect mitochondrial membrane from the peroxidation of cardiolipin and formation of mitochondrial permeability transition pores (mPTPs), to maintain the mitochondrial membrane potential, formation of adenosine triphosphate (ATP), and prevent cells form apoptosis [ 60 , 64 , 65 ]. That activity is supported by the activation of MT1R receptors located on the outer mitochondrial membrane.…”