Mammalian spermatozoa undergo rigorous dynamic changes during spermiogenesis, and fully mature sperms retain few mitochondria in the mid-piece. The most important function of mitochondria is the generation of chemical energy in the form of ATP and sperms use it to sustain their motility, which is one of the major factors to determine male fertility. Mitochondrial membrane potential (MMP) directly reflects the energy generation in terms of ATP. MMP of sperm can be measured by using tetra ethyl benzimidazolyl carbocyanine iodide (JC-1), a cationic dye that accumulates in energized mitochondria. At low concentration of ATP (generate low mitochondrial membrane potential), JC-1 is predominantly a monomer that yields green fluorescence with emission of 530±15nm. At high concentration of ATP (create high mitochondrial membrane potential), the dye aggregates yielding a red to orange colored emission (590±17.5nm). Therefore a low red/green fluorescence is indicative of depolarization whereas a high red/green ratio is indicative of hyperpolarization of mitochondria. Healthy mitochondria have high MMP and facilitate adequate synthesis of ATP to sustain the sperm motility.
The human semen consists of differentially motile subpopulations of spermatozoa, later could be separated through the swim up method. Usually, it had been observed that the MMP of swim-up sperms is higher than that of the crude samples followed by the leftover fraction.
Based on the observed correlation between sperm motility and MMP, some infertile patients’ sperm samples were assessed for their MMP and compared with normal sperms. We found that whenever the patients had compromised sperm motility (asthenozoospermia), they also showed lower MMP in comparison to those who had healthy sperms, clearly demonstrating the role of high MMP in sperm motility. MMP and motility of rodents, murine and human sperms were assessed concurrently and interestingly all the species showed that the MMP of their sperm samples reduced simultaneously with decreased motility as a function of time. Rat sperm MMP dropped more vigorously as compared to mouse and human sperms; may be because of the larger size of spermatozoa or their tails, and the required threshold MMP level would be higher to attain certain motility. Even though the MMP of sperms for different species cannot be compared with each other, it is the pattern of sperm MMP with respect to sperm motility, which has been focused in the present study.
To detect the effect of sperm motility inhibitors on sperm MMP in coherence with the reduction in motility; human sperm samples were treated with various spermicides, which made these sperms immotile or sluggish. Spermicides are known to impede the motility of the sperms but its effect on MMP has not been explored. We used known spermicides to inhibit the sperm motility and then its effect on MMP was monitored; their MMP level dropped tremendously after the treatment and strongly supported our hypothesis that MMP regulates sperm motility. To understand their correlation vice-versa; in another experiment, healthy motile sperms were treated with a known oxidative uncoupler (CCP) to encumber its MMP, which in turn reduced sperm motility remarkably. This experiment verified further that the sperm MMP regulates the sperm motility and directly associated with the health of spermatozoa. Finally, our results confirm the importance of mitochondrial membrane functionality in sperm motility.
Written by: Saurabh Kumar Agnihotri1, Ankit Kumar Agrawal1, Bilal Ahmad Hakim1, A L Vishwakarma2, T Narender3, Rekha Sachan4, Monika Sachdev1*
1Endocrinology Division, CSIR- Central Drug Research Institute, Lucknow, UP, India
2Sophisticated Analytical Instrument Facility, CSIR- Central Drug Research Institute, Lucknow, UP, India
3Medicinal and Process Chemistry Division, CSIR- Central Drug Research Institute, Lucknow, UP, India
4Department of Obstetrics and Gynaecology, King Georges’ Medical University, Lucknow, UP, India
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