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| Phospholipase C-Zeta Reveals the Underlying Pathological Influence after Density Gradient Centrifugation, Microfluidic Sorting, and Cryopreservation of Human Sperm |
| CAI Jing-jing |
| The Second Affiliated Hospital of Fujian Medical University, Quanzhou Fujian 362000, China |
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Abstract Objective: Sperm preparation techniques and cryopreservation are widely used in assisted reproductive techniques (ART). How to improve the quality of sperm management is a matter of great concern. Phospholipase C-zeta (PLCζ) is considered a sperm-specific agent that activates oocyte activation and thus playing a crucial role in male fertility. However, the potential mechanisms by which semen processing and cryopreservation on PLCζ contribute to keyhole have not been addressed. Methods: In this study, semen samples were taken from have not been addressed 10 normozoospermic men. Each semen sample was assigned to the following groups: density gradient centrifugation (DGC) as control, microfluidic sorting, and cryopreservation. Sperm parameters of molity, viability, membrane integrity, and intracellular ROS were evaluated during sperm preparation and cryopreservation. The expression of PLCζ in human sperm was determined by immunofluorescence and western blotting. Results: The results showed that molity, viability, and membrane integrity decreased in cryopreservation group. Intracellular ROS were also significantly increased compared to the the control group. There was no significant difference between DGC and microfluidic sorting group. Our investigation revealed that total levels of PLCζ were comparable between DGC and microfluidic sorting, but there were significantly reduced levels of PLCζ after cryopreservation as quantified by both immunofluorescenceand immunoblotting. PLCζ immunofluorescence in sperm revealed different PLCζ localization patterns around the acrosomal(Ac), equatorial (Eq), post-acrosomal (PA) areas of sperm heads, and their combination. The predominant patterns of PLCζ localization in DGC were similar to that of microfluidic sorting, with strong, with staining. In contrast, PLCζ staining in freeze-thawed sperm was considerably weaker fluorescence intensity. Conclusion: This study clarified the mechanism of sperm preparation and cryopreservation underlying effect on sperm characteristic, accompanied with PLCζ expresion. We demonstrated that microfluidic sorting provides a highly efficient preparation method for clinical selection of PLCζ-expressing sperm comparable to DGC gene expression. It is suggested that the cryopreservation of sperm has a significant detrimental effect on PLCζ.
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Received: 15 January 2023
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| Fund:The Science and Technology Projects of Quanzhou; grant number: 2019N085S; Startup Fund for Scientific Research, Fujian Medical University; grant number: 2018QH1100 |
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Corresponding Authors:
CAI Jing-jing. E-mail: caijjingjing@fjmu.edu.cn & caijj1983@163.com
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[1] Kashir J, Heynen A, Gadea J, et al.Effects of cryopres World Health Organization WHO laboratory manual for the examination and processing of human semen[M]. 5th ed. Geneva: World Health Organization, 2010.
[2] Quinn MM, Jalalian L, Ribeiro S, et al.Microfluidic sorting selects sperm for clinical with reduced DNA damage compared to density gradient centrifugation with swim-up in split semen samples[J]. Hum Reprod, 2018, 33(8): 1388-1393.
[3] Montagut M, Mieusset R, Isus F, Leandri R, et al.Sperm freezing to address the risk of azoospermia on the day of ICSI[J]. Hum Reprod, 2015,30(11):2486-2492.
[4] Hezavehei M, Sharafi M, Kouchesfahani HM, et al.Sperm cryopreservation: A review on current molecular cryobiology and advanced approaches[J]. Reprod Biomed Online, 2018,37(3):327-339.
[5] Zalata A, Hafez T, Comhaire F.Evaluation of the role of reactive oxygen species in male infertility[J]. Hum Reprod, 1995,10(6):1444-1451.
[6] Zini A, Finelli A, Phang D, et al.Influence of semen processing technique on human sperm DNA integrity[J]. Urology, 2000,56(6):1081-1084.
[7] Mahfouz RZ, du Plessis SS, Agarwal A, et al. Sperm viability, apoptosis, and intracellular reactive oxygen species levels in human spermatozoa before and after induction of oxidative stress[J]. Fertil Steril, 2010, 93(3): 814-821.
[8] Evenson DP, Larson KL, Jost LK.Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques[J]. J Androl, 2002, 23(1):25-43.
[9] Larson-Cook KL, Brannian JD, Hansen KA, et al.Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay[J]. Fertil Steril, 2003, 80(4):895-902.
[10] Bogle OA, Kumar K, Attardo-Parrinello C, et al.Identification of protein changes in human spermatozoa throughout the cryopreservation process[J]. Andrology, 2017, 5(1):10-22.
[11] Hezavehei M, Mirzaei M, Salekdeh GH, et al.Proteomics study reveals the molecular mechanisms underlying cryotolerance induced by mild sublethal stress in human sperm[J]. Cell Tissue Res, 2022, 387(1):143-157.
[12] Wang S, Wang W, Xu Y, et al.Proteomic characteristics of human sperm cryopreservation[J]. Proteomics, 2014, 14(2-3):298-310.
[13] Escoffier J, Yassine S, Hoi Chang Lee HC, et al. Subcellular localization of phospholipase Cζ in human sperm and its absence in DPY19L2-deficient sperm are consistent with its role in oocyte activation[J]. Mol Hum Reprod, 2015, 21(2):157-168.
[14] Yuan P, Yang C, Qiao J, et al.A novel homozygous mutation of phospholipase C zeta leading to defective human oocyte activation and fertilization failure[J]. Hum Reprod, 2020, 35(4):977-985.
[15] Kashir J, Heynen A, Coward K, et al.Effects of cryopreservation and density- gradient washing on phospholipase C zeta concentrations in human spermatozoa[J]. Reprod Biomed Online, 2011, 23(2):263-267.
[16] Moreau J, Fargeon S, Leandri RD, et al.Expression of phospholipase PLC Zeta in human spermatozoa: impact of cryopreservation[J]. Andrology, 2019, 7(3):315-318.
[17] Aitken, RJ, Curry BJ. Redox regulation of human sperm function: From the physiological control of sperm capacitation to the etiology of infertility and DNA damage in the germ line[J]. Antioxid Redox Signal, 2011,14(3):367-381.
[18] Dutta S, Majzoub A, Agarwal A.et al.Oxidative stress and sperm function: A systematic review on evaluation and management[J]. Arab J Urol, 2019, 17(2):87-97.
[19] Moazamian R, Polhemus A, Aitken RJ, et al.Oxidative stress and human spermatozoa: Diagnostic and functional signifificance of aldehydes generated as a result of lipid peroxidation[J]. Mol Hum Reprod, 2015, 21(6):502-515.
[20] Jayaraman V, Upadhya D, Narayan PK, et al.Sperm processing by swim-up and density gradient is effective in elimination of sperm with DNA damage[J]. J Assist Reprod Genet, 2012, 29(6):557-563.
[21] Malvezzi H, Sharma R, Agarwal A, et al.Sperm quality after density gradient centrifugation with three commercially available media: a controlled trial[J]. Reprod Biol Endocrinol, 2014, 12(121):121.
[22] Chi HJ, Kim JH, Ryu CS, et al.Protective effect of antioxidant supplementation in sperm-preparation medium against oxidative stress in human spermatozoa[J]. Hum Reprod, 2008, 23(5):1023-1028.
[23] Smith GD, Takayama S.Application of microfluidic technologies to human assisted reproduction[J]. Mol Hum Reprod, 2017, 23(4):257-268.
[24] Dai J, Zhang T, Lin G, et al.Homozygous pathogenic variants in ACTL9 cause fertilization failure and male infertility in humans and mice[J]. Am J Hum Genet, 2021, 108(3): 469-481.
[25] Park JH, Kim SK, Kim SH, et al.Relationship between phospholipase C zeta immunoreactivity and DNA fragmentation and oxidation in human sperm[J]. Obstet Gynecol Sci, 2015, 58(3): 232-238.
[26] Cho BS, Schuster TG, Zhu X, et al.Passively driven integrated microfluidic system for separation of motile sperm[J]. Anal Chem, 2003, 75(7): 1671-1675.
[27] Heytens E, Parrington J, De Sutter P, et al.Reduced amounts and abnormal forms of phospholipase C zeta (PLCzeta) in spermatozoa from infertile men[J]. Hum Reprod, 2009, 24(10): 2417-2428. |
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