Obstet Gynecol Sci.  2024 Sep;67(5):467-480. 10.5468/ogs.24019.

Phospholipase C zeta: a hidden face of sperm for oocyte activation and early embryonic development

• Azil S
• Mbaye MM
• Louanjli N
• Ghazi B
• Benkhalifa M

• KMID: 2559478
• DOI: http://doi.org/10.5468/ogs.24019

Abstract

Oocyte activation is a fundamental event in mammalian fertilization and is initiated by a cascade of calcium signaling and oscillation pathways. Phospholipase C zeta (PLCζ) is involved in modulating cortical granule exocytosis, releasing oocyte meiotic arrest, regulating gene expression, and early embryogenesis. These processes are considered to be initiated and controlled by PLCζ activity via the inositol-1,4,5-triphosphate pathway. The decrease or absence of functional PLCζ due to mutational defects in protein expression or maintenance can impair male fertility. In this literature review, we highlight the significance of PLCζ as a sperm factor involved in oocyte activation, its mechanism of action, the signaling pathway involved, and its close association with oocyte activation. Finally, we discuss the relationship between male infertility and PLCζ deficiency.

Keyword

Male infertility; Oocyte activation; Phospholipase C zeta; Calcium oscillations

Figure

• 

  Fig. 1. Human phospholipase C zeta (PLCζ) structure. (A) Modeling of human PLCζ homology (3D ribbon representation) [8]. The sequence of the protein is predicted using a model from the National Center for Biotechnology Information and the structure is predicted with the sequence AF-Q86YW0-F1 (National Center for Biotechnology Information, Bethesda, MD, USA) using the AlphaFold structure modeling and prediction tool. AlphaFold produces a confidence score per residue between 0 and 100 in the predicted local distance difference test (pLDDT). Some regions below 50 pLDDT may be unstructured in isolation. The PLCζ domain structure consists of four tandem EF hand domains at the N-terminus, the X and Y catalytic domains at the center of the molecule, and the C2 domain at the C-terminus. (B) Schematic representation of the distribution of different domains constituting PLCζ; the four tandem EF-hand domains at the N-terminus regulate the sensitivity of the protein to calcium levels, the second domain has the catalytic domains X and Y that cause changes in the functional ability of PLCζ to release Ca2+, and are separated by a short segment, the XY-linker, followed by a single C2 domain at the C-terminus that controls PLCζ function and it’s degree of sensitivity. 3D, three-dimensional.

• 

  Fig. 2. Schematic representation of the mechanisms of action of PAWP and PLCζ [10]. PAWP (left side) binds to the WW1 domain of the YAP protein; the activation of PLC gamma subsequently hydrolyzes PIP2 into a second messenger IP3; PLCζ (right side) induces the generation of IP3 through the hydrolysis of PIP2. The generated IP3 binds to IP3R on the ER and increases the levels of Ca2+ resulting in oocyte activation. GRAM, glucosyltransferases; PAWP, post-acrosomal WW1 domain binding protein; SF, sperm factor; PLCζ, phospholipase C zeta; YAP, yes-associated protein; PLCγ, phospholipase C gamma; IP3, inositol 1,4,5-triphosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; IP3R, inositol 1,4,5-trisphosphate receptor; ER, endoplasmic reticulum.

• 

  Fig. 3. Schematic representation of domains constituting PAWP [88]. SOAF, GRAM, Rab-like GTPase activators, and myotubularins domain. The post-acrosomal sheath WW domain-binding protein located in the perinuclear matrix of the sperm head shares a homology with the N-terminal half of the WW domain-binding protein 2; PY motifs with WW binding to the YGXPPX repeating motifs consensus binding site for group-1 WW domain-containing proteins followed by the N-terminal GRAM. PAWP, post-acrosomal sheath WW domain-binding protein; SOAF, sperm oocyte activating factor; GRAM, glucosyltransferases; PY, proline-tyrosine; GTPase, guanosine triphosphatase.

Reference

References

1. Bach PV, Schlegel PN. Male infertility. In : Strauss JF, Barbieri RL, editors. Yen and Jaffe’s reproductive endocrinology. 8th ed. Philadelphia: Elsevier;2019. p. 582–93.

2. Vander Borght M, Wyns C. Fertility and infertility: definition and epidemiology. Clin Biochem. 2018; 62:2–10.

3. McElreavey K, Krausz C, Patrat C, Fellous M. Male infertility and microdeletions of the Y chromosome. Gynecol Obstet Fertil. 2022; 30:405–12.

4. Khatun A, Rahman MS, Pang MG. Clinical assessment of the male fertility. Obstet Gynecol Sci. 2018; 61:179–91.

5. Saleh A, Kashir J, Thanassoulas A, Safieh-Garabedian B, Lai FA, Nomikos M. Essential role of sperm-specific PLCzeta in egg activation and male factor infertility: an update. Front Cell Dev Biol. 2020; 8:28.

6. Ramadan WM, Kashir J, Jones C, Coward K. Oocyte activation and phospholipase C zeta (PLCζ): diagnostic and therapeutic implications for assisted reproductive technology. Cell Commun Signal. 2012; 10:12.

7. Kashir J, Deguchi R, Jones C, Coward K, Stricker SA. Comparative biology of sperm factors and fertilizationinduced calcium signals across the animal kingdom. Mol Reprod Dev. 2013; 80:787–815.

8. Nomikos M, Swann K, Lai FA. Starting a new life: sperm PLC-zeta mobilizes the Ca²⁺ signal that induces egg activation and embryo development: an essential phospholipase C with implications for male infertility. Bioessays. 2012; 34:126–34.

9. Cox LJ, Larman MG, Saunders CM, Hashimoto K, Swann K, Lai FA. Sperm phospholipase Czeta from humans and cynomolgus monkeys triggers Ca²⁺ oscillations, activation and development of mouse oocytes. Reproduction. 2002; 124:611–23.

10. Nomikos M. Novel signalling mechanism and clinical applications of sperm-specific PLCζ. Biochem Soc Trans. 2015; 43:371–6.

11. Drevet JR. Fertilité — contraception mâles. Basic Clin Androl. 2009; 19:122–7.

12. Ravel C, Kazdar N, Drapier H, Duros S, Viard P. Assisted oocyte activation: a new tool for severe male factor infertility treatment. Med Sci (Paris). 2016; 32:198–203.

13. Torra-Massana M, Cornet-Bartolomé D, Barragán M, Durban M, Ferrer-Vaquer A, Zambelli F, et al. Novel phospholipase C zeta 1 mutations associated with fertilization failures after ICSI. Hum Reprod. 2019; 34:1494–504.

14. Swann K, Saunders CM, Rogers NT, Lai FA. PLCzeta (zeta): a sperm protein that triggers Ca²⁺ oscillations and egg activation in mammals. Semin Cell Dev Biol. 2006; 17:264–73.

15. Yelumalai S, Yeste M, Jones C, Amdani SN, Kashir J, Mounce G, et al. Total levels, localization patterns, and proportions of sperm exhibiting phospholipase C zeta are significantly correlated with fertilization rates after intracytoplasmic sperm injection. Fertil Steril. 2015; 104:561–8.e4.

16. Saunders CM, Larman MG, Parrington J, Cox LJ, Royse J, Blayney LM, et al. PLC zeta: a sperm-specific trigger of Ca (2+) oscillations in eggs and embryo development. Development. 2002; 129:3533–44.

17. Kashir J, Nomikos M, Lai FA. Phospholipase C zeta and calcium oscillations at fertilisation: the evidence, applications, and further questions. Adv Biol Regul. 2018; 67:148–62.

18. Nomikos M, Elgmati K, Theodoridou M, Calver BL, Nounesis G, Swann K, et al. Phospholipase Cζ binding to PtdIns (4,5)P2 requires the XY-linker region. J Cell Sci. 2011; 124:2582–90.

19. Nomikos M, Yu Y, Elgmati K, Theodoridou M, Campbell K, Vassilakopoulou V, et al. Phospholipase Cζ rescues failed oocyte activation in a prototype of male factor infertility. Fertil Steril. 2013; 99:76–85.

20. Yanagimachi R. Fertility of mammalian spermatozoa: its development and relativity. Zygote. 1994; 2:371–2.

21. Wu AT, Sutovsky P, Manandhar G, Xu W, Katayama M, Day BN, et al. PAWP, a sperm-specific WW domainbinding protein, promotes meiotic resumption and pronuclear development during fertilization. J Biol Chem. 2007; 282:12164–75.

22. Tavalaee M, Nasr-Esfahani MH. Expression profile of PLCζ, PAWP, and TR-KIT in association with fertilization potential, embryo development, and pregnancy outcomes in globozoospermic candidates for intra-cytoplasmic sperm injection and artificial oocyte activation. Andrology. 2016; 4:850–6.

23. PGrasa P, Coward K, Young C, Parrington J. The pattern of localization of the putative oocyte activation factor, phospholipase Czeta, in uncapacitated, capacitated, and ionophore-treated human spermatozoa. Hum Reprod. 2008; 23:2513–22.

24. Sutovsky P. New approaches to boar semen evaluation, processing and improvement. Reprod Domest Anim. 2015; 50 Suppl 2:11–9.

25. Kashir J, Nomikos M, Lai FA, Swann K. Sperm-induced Ca²⁺ release during egg activation in mammals. Biochem Biophys Res Commun. 2014; 450:1204–11.

26. Anifandis G, Michopoulos A, Daponte A, Chatzimeletiou K, Simopoulou M, Messini CI, et al. Artificial oocyte activation: physiological, pathophysiological and ethical aspects. Syst Biol Reprod Med. 2018; 65:3–11.

27. Kashir J, Heindryckx B, Jones C, De Sutter P, Parrington J, Coward K. Oocyte activation, phospholipase C zeta and human infertility. Hum Reprod Update. 2010; 16:690–703.

28. Yoda A, Oda S, Shikano T, Kouchi Z, Awaji T, Shirakawa H, et al. Ca²⁺ oscillation-inducing phospholipase C zeta expressed in mouse eggs is accumulated to the pronucleus during egg activation. Dev Biol. 2004; 268:245–57.

29. Kouchi Z, Shikano T, Nakamura Y, Shirakawa H, Fukami K, Miyazaki S. The role of EF-hand domains and C₂ domain in regulation of enzymatic activity of phospholipase Czeta. J Biol Chem. 2005; 280:21015–21.

30. Yoon SY, Jellerette T, Salicioni AM, Lee HC, Yoo MS, Coward K, et al. Human sperm devoid of PLC, zeta 1 fail to induce Ca(2+) release and are unable to initiate the first step of embryo development. J Clin Invest. 2008; 118:3671–81.

31. Jones KT. Mammalian sperm contain two factors for calcium release and egg activation: phospholipase C zeta and a cryptic activating factor. Mol Hum Reprod. 2018; 24:465–8.

32. Nozawa K, Satouh Y, Fujimoto T, Oji A, Ikawa M. Spermborne phospholipase C zeta-1 ensures monospermic fertilization in mice. Sci Rep. 2018; 8:1315.

33. Nomikos M, Blayney LM, Larman MG, Campbell K, Rossbach A, Saunders CM, et al. Role of phospholipase C-zeta domains in Ca²⁺-dependent phosphatidylinositol 4,5-bisphosphate hydrolysis and cytoplasmic Ca²⁺ oscillations. J Biol Chem. 2005; 280:31011–8.

34. Hachem A, Godwin J, Ruas M, Lee HC, Ferrer Buitrago M, Ardestani G, et al. PLCζ is the physiological trigger of the Ca²⁺ oscillations that induce embryogenesis in mammals but conception can occur in its absence. Development. 2017; 144:2914–24.

35. Jiménez-Movilla M, Hamze JG, Romar R. Oolemma receptors in mammalian molecular fertilization: function and new methods of study. Front Cell Dev Biol. 2021; 9:662032.

36. Escoffier J, Lee HC, Yassine S, Zouari R, Martinez G, Karaouzène T, et al. Homozygous mutation of PLCZ1 leads to defective human oocyte activation and infertility that is not rescued by the WW-binding protein PAWP. Hum Mol Genet. 2016; 25:878–91.

37. Yan Z, Fan Y, Wang F, Yan Z, Li M, Ouyang J, et al. Novel mutations in PLCZ1 cause male infertility due to fertilization failure or poor fertilization. Hum Reprod. 2020; 35:472–81.

38. Bunney TD, Katan M. PLC regulation: emerging pictures for molecular mechanisms. Trends Biochem Sci. 2011; 36:88–96.

39. Mu J, Zhang Z, Wu L, Fu J, Chen B, Yan Z, et al. The identification of novel mutations in PLCZ1 responsible for human fertilization failure and a therapeutic intervention by artificial oocyte activation. Mol Hum Reprod. 2020; 26:80–7.

40. Nomikos M, Sanders JR, Kashir J, Sanusi R, Buntwal L, Love D, et al. Functional disparity between human PAWP and PLCζ in the generation of Ca²⁺ oscillations for oocyte activation. Mol Hum Reprod. 2015; 21:702–10.

41. Dai J, Dai C, Guo J, Zheng W, Zhang T, Li Y, et al. Novel homozygous variations in PLCZ1 lead to poor or failed fertilization characterized by abnormal localization patterns of PLCζ in sperm. Clin Genet. 2020; 97:347–51.

42. Nasr-Esfahani MH, Deemeh MR, Tavalaee M. Artificial oocyte activation and intracytoplasmic sperm injection. Fertil Steril. 2010; 94:520–6.

43. Palermo GD, Neri QV, Schlegel PN, Rosenwaks Z. Intracytoplasmic sperm injection (ICSI) in extreme cases of male infertility. PLoS One. 2014; 9:e113671.

44. Aras-Tosun D, Cakar Z, Can A, Ozkavukcu S, Kaplanoglu I, Cinar O. Phospholipase C-zeta levels are not correlated with fertilisation rates in infertile couples. Andrologia. 2022; 54:e14269.

45. Ferrer-Vaquer A, Barragan M, Freour T, Vernaeve V, Vassena R. PLCζ sequence, protein levels, and distribution in human sperm do not correlate with semen characteristics and fertilization rates after ICSI. J Assist Reprod Genet. 2016; 33:747–56.

46. Kashir J, Konstantinidis M, Jones C, Heindryckx B, De Sutter P, Parrington J, et al. Characterization of two heterozygous mutations of the oocyte activation factor phospholipase C zeta (PLCζ) from an infertile man by use of minisequencing of individual sperm and expression in somatic cells. Fertil Steril. 2012; 98:423–31.

47. Kashir J, Konstantinidis M, Jones C, Lemmon B, Lee HC, Hamer R, et al. A maternally inherited autosomal point mutation in human phospholipase C zeta (PLCζ) leads to male infertility. Hum Reprod. 2012; 27:222–31.

48. Wang F, Zhang J, Kong S, Li C, Zhang Z, He X, et al. A homozygous nonsense mutation of PLCZ1 cause male infertility with oocyte activation deficiency. J Assist Reprod Genet. 2020; 37:821–8.

49. Taylor SL, Yoon SY, Morshedi MS, Lacey DR, Jellerette T, Fissore RA, et al. Complete globozoospermia associated with PLCζ deficiency treated with calcium ionophore and ICSI results in pregnancy. Reprod Biomed Online. 2010; 20:559–64.

50. Ferrer-Vaquer A, Barragan M, Freour T, Vernaeve V, Vassena R. PLCζ sequence, protein levels, and distribution in human sperm do not correlate with semen characteristics and fertilization rates after ICSI. J Assist Reprod Genet. 2016; 33:747–56.

51. Shimada K, Ono T, Mizushima S. Application of intracytoplasmic sperm injection (ICSI) for fertilization and development in birds. Gen Comp Endocrinol. 2014; 196:100–5.

52. Dale B, Wilding M, Coppola G, Tosti E. How do spermatozoa activate oocytes? Reprod Biomed Online. 2010; 21:1–3.

53. Fukami K, Inanobe S, Kanemaru K, Nakamura Y. Phospholipase C is a key enzyme regulating intracellular calcium and modulating the phosphoinositide balance. Prog Lipid Res. 2010; 49:429–37.

54. Meng X, Melo P, Jones C, Ross C, Mounce G, Turner K, et al. Use of phospholipase C zeta analysis to identify candidates for artificial oocyte activation: a case series of clinical pregnancies and a proposed algorithm for patient management. Fertil Steril. 2020; 114:163–74.

55. Neri QV, Lee B, Rosenwaks Z, Machaca K, Palermo GD. Understanding fertilization through intracytoplasmic sperm injection (ICSI). Cell Calcium. 2014; 55:24–37.

56. Vanden Meerschaut F, Leybaert L, Nikiforaki D, Qian C, Heindryckx B, De Sutter P. Diagnostic and prognostic value of calcium oscillatory pattern analysis for patients with ICSI fertilization failure. Hum Reprod. 2013; 28:87–98.

57. Ratti S, Follo MY, Ramazzotti G, Faenza I, Fiume R, Suh PG, et al. Nuclear phospholipase C isoenzyme imbalance leads to pathologies in brain, hematologic, neuromuscular, and fertility disorders. J Lipid Res. 2019; 60:312–7.

58. Yeste M, Jones C, Amdani SN, Yelumalai S, Mounce G, da Silva SJ, et al. Does advancing male age influence the expression levels and localisation patterns of phospholipase C zeta (PLCζ) in human sperm? Sci Rep. 2016; 6:27543.

59. Amdani SN, Yeste M, Jones C, Coward K. Sperm factors and oocyte activation: current controversies and considerations. Biol Reprod. 2015; 93:50.

60. Chithiwala ZH, Lee HC, Hill DL, Jellerette-Nolan T, Fissore R, Grow D, et al. Phospholipase C-zeta deficiency as a cause for repetitive oocyte fertilization failure during ovarian stimulation for in vitro fertilization with ICSI: a case report. J Assist Reprod Genet. 2015; 32:1415–9.

61. Alshahrani RA. Evaluation and characterization of PLCzeta in the male reproductive tract [Internet]. Riyadh: Alfaisal University;c2022. [cited 2024 Jul 1]. Available from: https://www.proquest.com/openview/50116e91037b1b3b0ad52c9b6f8afe85/1?pq-origsite=gscholar&cbl=2026366&diss=y.

62. Heytens E, Parrington J, Coward K, Young C, Lambrecht S, Yoon SY, et al. Reduced amounts and abnormal forms of phospholipase C zeta (PLCzeta) in spermatozoa from infertile men. Hum Reprod. 2009; 24:2417–28.

63. Jones C, Meng X, Coward K. Sperm factors and egg activation: phospholipase C zeta (PLCZ1) and the clinical diagnosis of oocyte activation deficiency. Reproduction. 2022; 164:F53–66.

64. Guo Y, Jiang J, Zhang H, Wen Y, Zhang H, Cui Y, et al. Proteomic analysis of dpy19l2-deficient human globozoospermia reveals multiple molecular defects. Proteomics Clin Appl. 2019; 13:e1900007.

65. Abdelhedi F, Chalas C, Petit JM, Abid N, Mokadem E, Hizem S, et al. Altered three-dimensional organization of sperm genome in DPY19L2-deficient globozoospermic patients. J Assist Reprod Genet. 2019; 36:69–77.

66. Koscinski I, Elinati E, Fossard C, Redin C, Muller J, Velez de la Calle J, et al. DPY19L2 deletion as a major cause of globozoospermia. Am J Hum Genet. 2011; 88:344–50.

67. Granados-Gonzalez V, Aknin-Seifer I, Touraine RL, Chouteau J, Wolf JP, Levy R. Preliminary study on the role of the human IZUMO gene in oocyte-spermatozoa fusion failure. Fertil Steril. 2008; 90:1246–8.

68. Cocco L, Follo MY, Manzoli L, Suh PG. Phosphoinositide-specific phospholipase C in health and disease. J Lipid Res. 2015; 56:1853–60.

69. Xu Z, Yao G, Niu W, Fan H, Ma X, Shi S, et al. Calcium Ionophore (A23187) rescues the activation of unfertilized oocytes after intracytoplasmic sperm injection and chromosome analysis of blastocyst after activation. Front Endocrinol (Lausanne). 2021; 12:692082.

70. Amdani SN, Yeste M, Jones C, Coward K. Phospholipase C zeta (PLCζ) and male infertility: clinical update and topical developments. Adv Biol Regul. 2016; 61:58–67.

71. Heindryckx B, De Gheselle S, Gerris J, Dhont M, De Sutter P. Efficiency of assisted oocyte activation as a solution for failed intracytoplasmic sperm injection. Reprod Biomed Online. 2008; 17:662–8.

72. Bonte D, Ferrer-Buitrago M, Dhaenens L, Popovic M, Thys V, De Croo I, et al. Assisted oocyte activation significantly increases fertilization and pregnancy outcome in patients with low and total failed fertilization after intracytoplasmic sperm injection: a 17-year retrospective study. Fertil Steril. 2019; 112:266–74.

73. Cardona Barberán A, Boel A, Vanden Meerschaut F, Stoop D, Heindryckx B. Diagnosis and treatment of male infertility-related fertilization failure. J Clin Med. 2020; 9:3899.

74. Javadian-Elyaderani S, Ghaedi K, Tavalaee M, Rabiee F, Deemeh MR, Nasr-Esfahani MH. Diagnosis of genetic defects through parallel assessment of PLCζ and CAPZA3 in infertile men with history of failed oocyte activation. Iran J Basic Med Sci. 2016; 19:281–9.

75. Campos G, Sciorio R, Esteves SC. Total fertilization failure after ICSI: insights into pathophysiology, diagnosis, and management through artificial oocyte activation. Hum Reprod Update. 2023; 29:369–94.

76. Ferrer-Buitrago M, Dhaenens L, Lu Y, Bonte D, Vanden Meerschaut F, De Sutter P, et al. Human oocyte calcium analysis predicts the response to assisted oocyte activation in patients experiencing fertilization failure after ICSI. Hum Reprod. 2018; 33:416–25.

77. Ebner T, Köster M, Shebl O, Moser M, Van der Ven H, Tews G, et al. Application of a ready-to-use calcium ionophore increases rates of fertilization and pregnancy in severe male factor infertility. Fertil Steril. 2012; 98:1432–7.

78. Vanden Meerschaut F, Nikiforaki D, Heindryckx B, De Sutter P. Assisted oocyte activation following ICSI fertilization failure. Reprod Biomed Online. 2014; 28:560–71.

79. Azad N, Nazarian H, Nazari L, Ghaffari Novin M, Piryaei A, Heidari MH, et al. Evaluation of PAWP and PLC? Expression in infertile men with previous ICSI fertilization failure. Urol J. 2018; 15:116–21.

80. Satouh Y, Nozawa K, Ikawa M. Sperm postacrosomal WW domain-binding protein is not required for mouse egg activation. Biol Reprod. 2015; 93:94.

81. Zafar MI, Lu S, Li H. Sperm-oocyte interplay: an overview of spermatozoon’s role in oocyte activation and current perspectives in diagnosis and fertility treatment. Cell Biosci. 2021; 11:4.

82. Tanhaei S, Abdali-Mashhadi S, Tavalaee M, JavadianElyaderani S, Ghaedi K, Seifati SM, et al. Assessment of sperm PAWP expression in infertile men. Urol J. 2019; 16:488–94.

83. Kamali-Dolat Abadi M, Tavalaee M, Shahverdi A, NasrEsfahani MH. Evaluation of PLCζ and PAWP expression in globozoospermic individuals. Cell J. 2016; 18:438–45.

84. Sutovsky P, Manandhar G, Wu A, Oko R. Interactions of sperm perinuclear theca with the oocyte: implications for oocyte activation, anti-polyspermy defense, and assisted reproduction. Microsc Res Tech. 2003; 61:362–78.

85. Nakai M, Ito J, Suyama A, Kageyama A, Tobari Y, Kashiwazaki N. Phospholipase Cζ (PLCζ) versus postacrosomal sheath WW domain-binding protein (PAWP): which molecule will survive as a sperm factor? Anim Sci J. 2020; 91:e13345.

86. Aarabi M, Qin Z, Xu W, Mewburn J, Oko R. Spermborne protein, PAWP, initiates zygotic development in Xenopus laevis by eliciting intracellular calcium release. Mol Reprod Dev. 2010; 77:249–56.

87. Aarabi M, Balakier H, Bashar S, Moskovtsev SI, Sutovsky P, Librach CL, et al. Sperm content of postacrosomal WW binding protein is related to fertilization outcomes in patients undergoing assisted reproductive technology. Fertil Steril. 2014; 102:440–7.

88. Kashir J, Nomikos M, Swann K, Lai FA. PLCζ or PAWP: revisiting the putative mammalian sperm factor that triggers egg activation and embryogenesis. Mol Hum Reprod. 2015; 21:383–8.

89. Gonzalez-Castro RA, Amoroso-Sanches F, Stokes JE, Graham JK, Carnevale EM. Localisation of phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (WBP2 N-terminal like) on equine spermatozoa and flow cytometry quantification of PLCZ1 and association with cleavage in vitro. Reprod Fertil Dev. 2019; 31:1778–92.

90. Nori W, Helmi ZR. Can follicular fluid 8-oxo-2'-deoxyguanosine predict the clinical outcomes in ICSI cycle among couples with normospermia male? Obstet Gynecol Sci. 2023; 66:430–40.