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. 2019 Aug 12;20(16):3921.
doi: 10.3390/ijms20163921.

Melatonin Improves the Fertilization Capacity of Sex-Sorted Bull Sperm by Inhibiting Apoptosis and Increasing Fertilization Capacitation via MT1

Chong-Yang Li  1 , Hai-Sheng Hao  1 , Ya-Han Zhao  1 , Pei-Pei Zhang  1 , Hao-Yu Wang  1 , Yun-Wei Pang  1 , Wei-Hua Du  1 , Shan-Jiang Zhao  1 , Yan Liu  1 , Jin-Ming Huang  2 , Jing-Jing Wang  1 , Wei-Min Ruan  3 , Tong Hao  1 , Russel J Reiter  4 , Hua-Bin Zhu  5 , Xue-Ming Zhao  6
Affiliations

Melatonin Improves the Fertilization Capacity of Sex-Sorted Bull Sperm by Inhibiting Apoptosis and Increasing Fertilization Capacitation via MT1

Chong-Yang Li et al. Int J Mol Sci. .

Abstract

Little information is available regarding the effect of melatonin on the quality and fertilization capability of sex-sorted bull sperm, and even less about the associated mechanism. Sex-sorted sperm from three individual bulls were washed twice in wash medium and incubated in a fertilization medium for 1.5 h, and each was supplemented with melatonin (0, 10-3 M, 10-5 M, 10-7 M, and 10-9 M). The reactive oxygen species (ROS) and endogenous antioxidant activity (glutathione peroxidase (GPx); superoxide dismutase (SOD); catalase (CAT)), apoptosis (phosphatidylserine [PS] externalization; mitochondrial membrane potential (Δψm)), acrosomal integrity events (malondialdehyde (MDA) level; acrosomal integrity), capacitation (calcium ion [Ca2+]i level; cyclic adenosine monophosphate (cAMP); capacitation level), and fertilization ability of the sperm were assessed. Melatonin receptor 1 (MT1) and 2 (MT2) expression were examined to investigate the involvement of melatonin receptors on sex-sorted bull sperm capacitation. Our results show that treatment with 10-5 M melatonin significantly decreased the ROS level and increased the GPx, SOD, and CAT activities of sex-sorted bull sperm, which inhibited PS externalization and MDA levels, and improved Δψm, acrosomal integrity, and fertilization ability. Further experiments showed that melatonin regulates sperm capacitation via MT1. These findings contribute to improving the fertilization capacity of sex-sorted bull sperm and exploring the associated mechanism.

Keywords: bull; fertilization; melatonin; sex-sorted; sperm.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effect of melatonin on ROS level and CAT, GPx, and SOD activities in sex-sorted bull sperm. (A) ROS staining. Scale bar = 20 μm. (B) a,b,c,d Values with no common superscripts represent statistical significance (p < 0.05), the same as below. (C) Effect of melatonin on the level of CAT, GPx, and SOD activities in sex-sorted bull sperm.
Figure 2
Figure 2
Effect of melatonin on the PS externalization events of sex-sorted bull sperm. (A) Negative control. (B) PI staining control. (C) Annexin V FITC staining control. (D) Analysis of sex-sorted bull sperm. Quadrants represented viable sperm (lower-left quadrant), early apoptotic sperm (lower-right quadrant), early necrotic sperm (upper-right quadrant), and necrotic sperm (upper-left quadrant). (E) Effect of melatonin on the PS externalization events of sex-sorted bull sperm. Lowercase letters represent statistical significant difference (p < 0.05).
Figure 3
Figure 3
Effect of melatonin on the level of Δψm of sex-sorted bull sperm (A) Negative control. (B) Positive control. (C) Analysis of sex-sorted bull sperm. Quadrants represented low Δψm sperm (lower-right quadrant) and high Δψm sperm (upper-right quadrant). (D) Effect of melatonin on the level of Δψm of sex-sorted bull sperm. Lowercase letters represent statistical significant difference (p < 0.05).
Figure 4
Figure 4
Effect of melatonin on the level of MDA of sex-sorted bull sperm. Lowercase letters represent statistical significant difference (p < 0.05).
Figure 5
Figure 5
Effect of melatonin on the acrosome integrity of sex-sorted bull sperm. (A) Negative control. (B) PI staining. (C) FITC-PNA staining. (D) FITC-PNA/PI staining. Acrosome staining of sex-sorted sperm of bull. Quadrants represented viable sperm with integral acrosome (lower-left quadrant), viable sperm with damaged acrosome (lower-right quadrant), dead sperm with integral acrosome (upper-left quadrant), and dead sperm with damaged acrosomes (upper-right quadrant). (E) Effect of melatonin on the acrosome integrity of sex-sorted bull sperm. Lowercase letters represent statistical significant difference (p < 0.05).
Figure 6
Figure 6
Effect of melatonin on the level of [Ca2+]i and cAMP of sex-sorted bull sperm. (A) Effect of melatonin on the level of [Ca2+]i of sex-sorted bull sperm. (B) Effect of melatonin on the level of cAMP of sex-sorted bull sperm. Lowercase letters represent statistical significant difference (p < 0.05).
Figure 7
Figure 7
Effect of melatonin on sex-sorted bull sperm capacitation. (A) Representative image of 0 M group. (B) Representative image of 10−3 M group. (C) Representative image of 10−5 M sperm analysis. (D) Representative image of 10−7 M group. (E) Representative image of 10−9 M sperm analysis. The quadrants represented viable sperm without destabilized membranes (YoPro− and M540−), viable sperm with destabilized membranes (YoPro− and M540+), nonviable sperm without destabilized membranes (YoPro− and M540+), and nonviable sperm with destabilized membranes (YoPro+ and M540+). (F) Effect of melatonin on sex-sorted bull sperm capacitation. Lowercase letters represent statistical significant difference (p < 0.05).
Figure 8
Figure 8
Effect of 4-P-POD and luzindole on the level of [Ca2+]i, cAMP, and capacitation of sex-sorted bull sperm. (A) Western blot image of MT1 and MT2 protein expression. (B) Effect of 4-P-POD and luzindole on the level of [Ca2+]i of sex-sorted bull sperm. (C) Effect of 4-P-POD and luzindole on the level of cAMP of sex-sorted bull sperm. (D) Effect of 4-P-POD and luzindole on the level of capacitation of sex-sorted bull sperm. Lowercase letters represent statistical significant difference (p < 0.05).
Figure 9
Figure 9
Effect of melatonin on the IVF efficiency of sex-sorted bull sperm. Lowercase letters represent statistical significant difference (p < 0.05).
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References

    1. Barceló-Fimbres M., Campos-Chillón L.F., Seidel G.E., Jr. In vitro fertilization using non-sexed and sexed bovine sperm: Sperm concentration, sorter pressure, and bull effects. Reprod. Domest. Anim. 2011;46:495–502. doi: 10.1111/j.1439-0531.2010.01696.x. - DOI - PubMed
    1. Leahy T., Celi P., Bathgate R., Evans G., Maxwell W.M., Marti J.I. Flow-sorted ram spermatozoa are highly susceptible to hydrogen peroxide damage but are protected by seminal plasma and catalase. Reprod. Fertil. Dev. 2010;22:1131–1140. doi: 10.1071/RD09286. - DOI - PubMed
    1. Balao da Silva C.M., Ortega-Ferrusola C., Morrell J.M., Rodriguez Martínez H., Peña F.J. Flow Cytometric chromosomal sex sorting of stallion spermatozoa induces oxidative stress on mitochondria and genomic DNA. Reprod. Domest. Anim. 2016;51:18–25. doi: 10.1111/rda.12640. - DOI - PubMed
    1. Li C.Y., Zhao Y.H., Hao H.S., Wang H.Y., Huang J.M., Yan C.L., Du W.H., Pang Y.W., Zhang P.P., Liu Y., et al. Resveratrol significantly improves the fertilisation capacity of bovine sex-sorted semen by inhibiting apoptosis and lipid peroxidation. Sci. Rep. 2018;8:7603. doi: 10.1038/s41598-018-25687-z. - DOI - PMC - PubMed
    1. Plaza Davila M., Martin Munoz P., Tapia J.A., Ortega Ferrusola C., Balao da Silva C.C., Peña F.J. Inhibition of mitochondrial complex i leads to decreased motility and membrane integrity related to increased hydrogen peroxide and reduced ATP production, while the inhibition of glycolysis has less impact on sperm motility. PLoS ONE. 2015;10:e0138777. doi: 10.1371/journal.pone.0138777. - DOI - PMC - PubMed

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