Melatonin rescues impaired penetration ability of human spermatozoa induced by mitochondrial dysfunction

in Reproduction
Correspondence should be addressed to H-F Huang; Email: huanghefg@hotmail.com

*(X-Y Zhang and Y-M Xiong contributed equally to this work)

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Fertilization failure often occurs during in vitro fertilization (IVF) cycles despite apparently normal sperm and oocytes. Accumulating evidence suggests that mitochondria play crucial roles in the regulation of sperm function and male fertility. 3-Nitrophthalic acid (3-NPA) can induce oxidative stress in mitochondria, and melatonin, as an antioxidant, can improve mitochondrial function by reducing mitochondrial oxidative stress. The role of sperm mitochondrial dysfunction in fertilization failure during IVF is unclear. The present study revealed that spermatozoa with low, or poor, fertilization rates had swollen mitochondria, increased mitochondria-derived ROS, and attenuated mitochondrial respiratory capacity. 3-NPA treatment enhanced mitochondrial dysfunction in sperm. Spermatozoa with poor fertilization rates, and spermatozoa treated with 3-NPA, had reduced penetration ability. The concentration of melatonin was decreased in semen samples with low and poor fertilization rates. Melatonin, not only decreased excessive mitochondria-derived ROS, but also ‘rescued’ the reduced penetration capacity of spermatozoa treated with 3-NPA. Taken together, the study suggested that mitochondria-derived ROS and mitochondrial respiratory capacity are independent bio-markers for sperm dysfunction, and melatonin may be useful in improving sperm quality and overall male fertility.

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  • Supplementary Table 1. Parameter settings used with the Hamilton-Thorne CASA instrument (software version 12)

 

    Society for Reproduction and Fertility

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    Impaired mitochondrial ultra-structure and increased mitochondria-derived ROS are observed in sperm of unexplained infertile male patients. (A) Mitochondrial ultra-structures in sperm from FR1, FR2 and FR3 groups were observed by TEM. The red arrows indicate impaired mitochondrial ultra-structure (such as metamorphism, black bulk, physalides and decreased cristae) in sperm from group FR2 and FR3. Four samples in each group (FR1, 2 and 3), and 20–40 sperm in each sample, were analyzed. Scale bar = 0.2 μm. (B) The distribution and content of mitochondria-derived ROS in sperm from FR1, FR2 and FR3 groups were analyzed by MitoSOX staining and fluorescence microscopy. Red color indicates MitoSOX staining. The middle and lower panels showed the overlapped pictures of fluorescence and bright fields. Scale bar = 50 μm in the upper and middle panels, scale bar = 5 μm in the lower panel. (C) The MFI of MitoSOX staining in sperm from FR1, FR2 and FR3 groups in the absence or presence of 0.3 mM melatonin was assessed by flow cytometry and shown as bar graph (mean ± s.e.m.). *, ** and ***, P < 0.05, P < 0.01 and P < 0.001, respectively, determined by ANOVA with the Tukey’s post hoc test. Data are representative of three independent experiments. (D) Mitochondrial mass in sperm was assessed by Mitotracker Green FM staining and flow cytometry and shown as bar graph (mean ± s.e.m.). n, number of samples. Data are representative of three independent experiments.

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    Mitochondrial respiratory capacity is reduced in sperm of FR2 and FR3 groups compared to that of FR1 group. (A) Mitochondrial respiratory curves of sperm were assessed with Seahorse system. ‘*’ or ‘**’ indicates significant difference when compared FR3 with FR1, P values were determined by the Student t test. (B) Basal OCR, oligomycin-induced OCR, FCCP-induced OCR and rotenone plus antimycin-induced OCR of mitochondria in sperm from FR1, FR2 and FR3 groups were shown as bar graph (mean ± s.e.m.). P values were determined by ANOVA with the Tukey’s post hoc test. * and **, P < 0.05 and P < 0.01, respectively. n, number of samples. Data are representative of three independent experiments.

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    Effects of 3-NPA on mitochondrial respiratory capacity, mitochondria-derived ROS and kinematic parameters of sperm with normal IVF rate from FR1 group. (A) Mitochondrial respiratory curves were assessed by Seahorse system for sperm treated without or with 3-NPA (0.5 mM or 1 mM), respectively. ‘*’, ‘**’ or ‘***’ indicates significant differences when compared 3-NPA 1 mM group with 3-NPA 0 mM group. P values were determined by the Student t test. (B) Basal OCR, oligomycin-induced OCR, FCCP-induced OCR and rotenone plus antimycin-induced OCR of sperm in the absence or presence of 3-NPA (0.5 mM or 1 mM) were shown as bar graph (mean ± s.e.m.). P values were determined by ANOVA with the Tukey’s post hoc test. (C) The MFI of MitoSOX staining in sperm treated without or with 3-NPA (0.5 mM or 1 mM) in the absence or presence of 0.3 mM melatonin was assessed by flow cytometry and shown as bar graph (mean ± s.e.m.). P values were determined by ANOVA with the Tukey’s post hoc test. (D, E, F, G, H, I, J, K, L and M) The kinematic parameters of sperm treated without or with 3-NPA (0.5 mM or 1 mM) in the absence or presence of 0.3 mM melatonin were assessed by CASA and shown as bar graph (mean ± s.e.m.). P values were determined by ANOVA with the Tukey’s post hoc test. *, ** and *** P < 0.05, P < 0.01 and P < 0.001, respectively. n, number of samples. Data are representative of three independent experiments.

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    Melatonin rescues the impaired penetration potential of sperm treated with 3-NPA or sperm with poor IVF rate. (A) Melatonin concentration in seminal plasma samples from FR1, FR2 and FR3 groups was assessed by ELISA and shown as bar graph (mean ± s.e.m.). P values were determined by the Student t test. n, number of samples. (B) The penetration of hamster zona-free oocytes by sperm treated without or with 3-NPA (0.5 mM or 1 mM) in the absence or presence of 0.3 mM melatonin were observed by confocal fluorescence microscopy. Green color indicates wheat germ agglutinin (WGA) staining of the membrane of the oocyte. Blue color indicates DAPI staining of the nucleus. The red arrow indicates the nucleus of the oocyte, the other DAPI + nucleus indicate the nucleus of sperm that entered into the oocyte. Scale bar = 20 μm. (C) The penetration index of sperm treated without or with 3-NPA (0.5 mM or 1 mM) in the absence or presence of 0.3 mM melatonin was shown as bar graph (mean ± s.e.m.). P values were determined by ANOVA with the Tukey’s post hoc test. (D) The penetration of hamster zona-free oocytes by sperm with poor IVF rate in the absence or presence of melatonin (0.3 mM or 0.5 mM) was observed by confocal fluorescence microscopy. Green color indicates WGA staining of the membrane of the oocyte. Blue color indicates DAPI staining of the nucleus. The red arrow indicates the nucleus of the oocyte, the other DAPI+ nucleus indicate the nucleus of sperm that entered the oocyte. (E) The penetration index of sperm with poor IVF rates, in the absence or presence of melatonin (0.3 mM or 0.5 mM) shown as bar graph (mean ± s.e.m.). P values were determined by ANOVA with the Tukey’s post hoc test. *, ** and ***, P < 0.05, P < 0.01 and P < 0.001, respectively. n, number of oocytes. Data are representive of three independent experiments.

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