Assessment of Expression Levels and Localization Patterns of Phospholipase C zeta in Different Grades of HOST in Human Sperm

Document Type : Original Article


1 Department of Anatomy, Faculty of Medicine, Kurdistan University of Medical Sciences, Sannandaj, Iran

2 Department of Midwifery, Falavarjan Branch, Islamic Azad University, Isfahan, Iran


Background: Phospholipase C zeta (PLC-ζ) deficiency in sperm can underlie oocyte activation failure after intracytoplasmic
sperm injection (ICSI). The aim of this study was to determine PLC-ζ expression and location in individual
spermatozoa in each host score so that a hypo-osmotic swelling test (HOST) may be used to help routine sperm selection
for ICSI.
Materials and Methods: In this experimental study, fresh semen samples were randomly obtained from 30 men who
were referred to the Andrology Unit of the Infertility Center. Samples were processed by density gradient centrifugation
(DGC) and exposed to hypotonic conditions. Seven different tail patterns, classified from ‘a’ to ‘g’ can be detected
according to World Health Organization (WHO) criteria. Then, the PLC-ζ protein localization pattern was assessed by
quantitative Immunofluorescence in individual sperm Host grades. Moreover, the sperm content of PLC-ζ protein was
evaluated by flow cytometry correlated with semen analysis parameters.
Results: In the present study, quantitive immunofluorescence analysis indicated that sperm from different host grades
exhibited seven localization patterns of PLC-ζ of acrosomal (A); equatorial (EQ), and postacrosomal (PA) patterns.
A+EQ=acrosomal and equatorial, A+PA=acrosomal and post-acrosomal, EQ+PA=equatorial and post-crosomal, and
The sperm from HOST grade 'd' exhibited significantly higher PLC-ζ (A+PA) and (A+EQ+PA) staining compared to sperm
from other grades (P=0.006). The sperm from grade 'd' exhibited higher PLC-ζ (EQ+PA) compared with other grades
(P=0.001). However, grade 'd' was not significantly different from 'c' (P=0.087). Analysis of the combined results confirmed
that there was a clear reduction in PLC-ζ immunofluorescence in Host grades 'a', 'f' and 'g' sperms.
Conclusion: Our data suggest that HOST may represent a useful diagnostic tool for the selection of sperms exhibiting
a higher level of PLC-ζ expression.


Main Subjects

1. Lee SH, Lee JH, Park YS, Yang KM, Lim CK. Comparison of clini­cal outcomes between in vitro fertilization (IVF) and intracytoplas­mic sperm injection (ICSI) in IVF-ICSI split insemination cycles. Clin Exp Reprod Med. 2017; 44(2): 96-104.
2. 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(12): 3899.
3. 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(1): F53-F66.
4. Wang M, Zhu L, Liu C, He H, Wang C, Xing C, et al. A novel assist­ed oocyte activation method improves fertilization in patients with recurrent fertilization failure. Front Cell Dev Biol. 2021; 9: 672081.
5. Tavalaee M, Nomikos M, Lai FA, Nasr-Esfahani MH. Expression of sperm PLCζ and clinical outcomes of ICSI-AOA in men affected by globozoospermia due to DPY19L2 deletion. Reprod Biomed On­line. 2018; 36(3): 348-355.
6. Haghighat S, Tavalaee M, Kouhkan A, Zakeri Z, Noureddini M, Shahverdi AH, et al. Reduction of truncated kit expression in men with abnormal semen parameters, globozoospermia and history of low or fertilization failure. Cell J. 2019; 21(3): 314-321.
7. Janghorban-Laricheh E, Ghazavi-Khorasgani N, Tavalaee M, Zohrabi D, Abbasi H, Nasr-Esfahani MH. An association between sperm PLCζ levels and varicocele? J Assist Reprod Genet. 2016; 33(12): 1649-1655.
8. Tavalaee M, Kiani-Esfahani A, Nasr-Esfahani MH. Relationship be­tween phospholipase C-zeta, semen parameters, and chromatin status. Syst Biol Reprod Med. 2017; 63(4): 259-268.
9. Saleh A, Kashir J, Thanassoulas A, Safieh-Garabedian B, Lai FA, Nomikos M. Essential role of sperm-specific PLC-Zeta in egg ac­tivation and male factor infertility: an update. Front Cell Dev Biol. 2020; 8: 28.
10. Kumar N, Singh AK. Semen quality parameters in male partners of infertile couples and their correlation with socio-demographic fea­tures in rural tertiary care center of southern india: an observational study. J Infertil Reprod Biol. 2022; 10(1): 1-6.
11. 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(6): 747-756.
12. Grasa 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(11): 2513-2522.
13. Kashir J, Jones C, Mounce G, Ramadan WM, Lemmon B, Hein­dryckx B, et al. Variance in total levels of phospholipase C zeta (PLC-ζ) in human sperm may limit the applicability of quantitative immunofluorescent analysis as a diagnostic indicator of oocyte ac­tivation capability. Fertil Steril. 2013; 99(1): 107-117. e3.
14. 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(3): 561-568. e4.
15. 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(9): 1415-1419.
16. Lee HC, Arny M, Grow D, Dumesic D, Fissore RA, Jellerette-Nolan T. Protein phospholipase C Zeta1 expression in patients with failed ICSI but with normal sperm parameters. J Assist Reprod Genet. 2014; 31(6): 749-756.
17. Bassiri F, Tavalaee M, Shiravi AH, Mansouri S, Nasr-Esfahani MH. Is there an association between HOST grades and sperm quality? Hum Reprod. 2012; 27(8): 2277-2284.
18. Stanger JD, Vo L, Yovich JL, Almahbobi G. Hypo-osmotic swelling test identifies individual spermatozoa with minimal DNA fragmenta­tion. Reprod Biomed Online. 2010; 21(4): 474-484.
19. Lu JC, Huang YF, Lü NQ. WHO laboratory manual for the examination and processing of human semen: its applicability to andrology labora­tories in China. Zhonghua Nan Ke Xue. 2010; 16(10): 867-871.
20. Bassiri F, Tavalaee M, Nasr Esfahani MH. Correlation between different patterns of hypo-osmotic swelling and sperm functional tests. Int J Fertil Steril. 2013; 7(3): 193-198.
21. Wildy ML, Boyd L, Fourie J, Makoyo O, Huyser C. The develop­ment of a simplified swim-up method for sperm processing. J Infer­til Reprod Biol. 2021; 9 (4): 160-167.
22. Azad N, Nazarian H, Ghaffari Novin M, Masteri Farahani R, Piryaei A, Heidari MH. Phospholipase C zeta parameters in sperm from polymorphic teratozoospermic men. Ann Anat. 2018; 215: 63-70.
23. Hermann AJE, Sylvère NKG, Guillaume YY, Amadou CF, Marie- Florence N, Alexis BG, et al. The asthenozoospermia as a major sperm abnormality of people received in Institute Pasteur of Cote d'Ivoire for a semen analysis. J Infertil Reprod Biol. 2019; 7(3): 17- 20.
24. Penitente-Filho JM, Dias JCO, Oliveira FA, Silveira CO, Torres CAA. Correlation between sperm motility and hypoosmotic swell­ing test on cryopreserved goat semen. Magistra. 2015; 27(3/4): 468-473.
25. Pang MG, You YA, Park YJ, Oh SA, Kim DS, Kim YJ. Numerical chromosome abnormalities are associated with sperm tail swelling patterns. Fertil Steril. 2010; 94(3): 1012-1020.
26. Jeyendran RS, Van der Ven HH, Perez-Pelaez M, Crabo BG, Zan­eveld LJ. Development of an assay to assess the functional integ­rity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil. 1984; 70(1): 219-228.
27. Ozdemir U, Atayurt Z, Sik A, Ozdamar O, Aba YA, Ozolcay O. Can hypo-osmotic swelling test (host) improve pregnancy outcomes in unexplained iinfertility patients with normal semen parameters un­dergoing ICSI-frozen embryo transfer cycles? Clin Exp Health Sci. 2021; 11(3): 536-542.
28. Hossain A, Osuamkpe C, Hossain S, Phelps JY. Spontaneously developed tail swellings (SDTS) influence the accuracy of the hy­po-osmotic swelling test (HOS-test) in determining membrane in­tegrity and viability of human spermatozoa. J Assist Reprod Genet. 2010; 27(2-3): 83-86.
29. Kashir J, Mistry BV, BuSaleh L, Abu-Dawas R, Nomikos M, Ajlan A, et al. Phospholipase C zeta profiles are indicative of optimal sperm parameters and fertilisation success in patients undergoing fertility treatment. Andrology. 2020; 8(5): 1143-1159.
30. 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(10): 2417-2428.
31. 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(1): 222-231.
32. 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(1): e14269.
33. 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(2): 440-447.
34. 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(5): 878-891.
35. 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(5): 611-623.
36. Morrell JM, Alsina MS, Abraham MC, Sjunnesson Y. Practical ap­plications of sperm selection techniques for improving reproduction efficiency. Anim Reprod. 2016; 13(3): 340-345.
37. Kashir J, Heynen A, Jones C, Durrans C, Craig J, Gadea J, et al. Effects of cryopreservation and density-gradient washing on phos­pholipase C zeta concentrations in human spermatozoa. Reprod Biomed Online. 2011; 23(2): 263-267.
38. Park JH, Kim SK, Kim J, Kim JH, Chang JH, Jee BC, et al. Rela­tionship between phospholipase C zeta immunoreactivity and DNA fragmentation and oxidation in human sperm. Obstet Gynecol Sci. 2015; 58(3): 232-238.
39. Tavalaee M, Kiani-Esfahani A, Nasr-Esfahani MH. Relationship between potential sperm factors involved in oocyte activation and sperm DNA fragmentation with intra-cytoplasmic sperm injection clinical outcomes. Cell J. 2017; 18(4): 588-596.
40. Bhattacharya SM. Hypo-osmotic swelling test and unexplained re­peat early pregnancy loss. J Obstet Gynaecol Res. 2010; 36(1): 119-122.