Advertisement

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

      Objective

      To examine whether similar levels of phospholipase C zeta (PLC-ζ) protein are present in sperm from men whose ejaculates resulted in normal oocyte activation, and to examine whether a predominant pattern of PLC-ζ localization is linked to normal oocyte activation ability.

      Design

      Laboratory study.

      Setting

      University laboratory.

      Patient(s)

      Control subjects (men with proven oocyte activation capacity; n = 16) and men whose sperm resulted in recurrent intracytoplasmic sperm injection failure (oocyte activation deficient [OAD]; n = 5).

      Intervention(s)

      Quantitative immunofluorescent analysis of PLC-ζ protein in human sperm.

      Main Outcome Measure(s)

      Total levels of PLC-ζ fluorescence, proportions of sperm exhibiting PLC-ζ immunoreactivity, and proportions of PLC-ζ localization patterns in sperm from control and OAD men.

      Result(s)

      Sperm from control subjects presented a significantly higher proportion of sperm exhibiting PLC-ζ immunofluorescence compared with infertile men diagnosed with OAD (82.6% and 27.4%, respectively). Total levels of PLC-ζ in sperm from individual control and OAD patients exhibited significant variance, with sperm from 10 out of 16 (62.5%) exhibiting levels similar to OAD samples. Predominant PLC-ζ localization patterns varied between control and OAD samples with no predictable or consistent pattern.

      Conclusion(s)

      The results indicate that sperm from control men exhibited significant variance in total levels of PLC-ζ protein, as well as significant variance in the predominant localization pattern. Such variance may hinder the diagnostic application of quantitative PLC-ζ immunofluorescent analysis.

      Key Words

      To read this article in full you will need to make a payment

      Subscribe:

      Subscribe to Fertility and Sterility
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect

      References

        • Stricker S.A.
        Comparative biology of calcium signalling during fertilisation and egg activation in mammals.
        Dev Biol. 1999; 211: 157-176
        • Whitaker M.
        Calcium at fertilization and in early development.
        Physiol Rev. 2006; 86: 25-88
        • Berridge M.J.
        Inositol trisphosphate and calcium signalling mechanisms.
        Biochim Biophys Acta. 2009; 1793: 933-940
        • Jones K.T.
        Mammalian egg activation: from Ca2+ spiking to cell cycle progression.
        Reproduction. 2005; 130: 813-823
        • Dozortsev D.
        • Qian C.
        • Ermilov A.
        • Rybouchkin A.
        • De Sutter P.
        • Dhont M.
        Sperm-associated oocyte-activating factor is released from the spermatozoon within 30 minutes after injection as a result of the sperm-oocyte interaction.
        Hum Reprod. 1997; 12: 2792-2796
        • Swann K.
        • Saunders C.M.
        • Rogers N.T.
        • Lai F.A.
        PLCzeta (zeta): a sperm protein that triggers Ca2+ oscillations and egg activation in mammals.
        Semin Cell Dev Biol. 2006; 17: 264-273
        • Parrington J.
        • Davis L.C.
        • Galione A.
        • Wessel G.
        Flipping the switch: how a sperm activates the egg at fertilization.
        Dev Dyn. 2007; 236: 2027-2038
        • 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
        • Ito J.
        • Parrington J.
        • Fissore R.A.
        PLCζ and its role as a trigger of development in vertebrates.
        Mol Reprod Dev. 2011; 78: 846-853
        • Cox L.J.
        • Larman M.G.
        • Saunders C.M.
        • Hashimoto K.
        • Swann K.
        • Lai F.A.
        Sperm phospholipase C zeta from humans and cynomolgus monkeys triggers Ca2+ oscillations, activation and development of mouse oocytes.
        Reproduction. 2002; 124: 611-623
        • Saunders C.M.
        • Larman M.G.
        • Parrington J.
        • Cox L.J.
        • Royse J.
        • Blayney L.M.
        • et al.
        PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development.
        Development. 2002; 129: 3533-3544
        • Saunders C.M.
        • Swann K.
        • Lai F.A.
        PLC zeta, a sperm-specific PLC and its potential role in fertilization.
        Biochem Soc Symp. 2007; 74: 23-36
        • Kashir J.
        • Jones C.
        • Coward K.
        Calcium Oscillations, oocyte activation, and phospholipase C zeta.
        Adv Exp Med Biol. 2012; 740: 1095-1121
        • 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: 21015-21021
        • Fujimoto S.
        • Yoshida N.
        • Fukui T.
        • Amanai M.
        • Isobe T.
        • Itagaki C.
        • et al.
        Mammalian phospholipase Czeta induces oocyte activation from the sperm perinuclear matrix.
        Dev Biol. 2004; 274: 370-383
        • Kurokawa M.
        • Sato K.
        • Wu H.
        • He C.
        • Malcuit C.
        • Black S.J.
        • et al.
        Functional, biochemical, and chromatographic characterization of the complete [Ca2+]i oscillation–inducing activity of porcine sperm.
        Dev Biol. 2005; 282: 376-392
        • Knott J.G.
        • Kurokawa M.
        • Fissore R.A.
        • Schultz R.M.
        • Williams C.J.
        Transgenic RNA interference reveals role for mouse sperm phospholipase Czeta in triggering Ca2+ oscillations during fertilization.
        Biol Reprod. 2005; 72: 992-996
        • Bedford-Guaus S.J.
        • Yoon S.Y.
        • Fissore R.A.
        • Choi Y.H.
        • Hinrichs K.
        Microinjection of mouse phospholipase C zeta complementary RNA into mare oocytes induces long-lasting intracellular calcium oscillations and embryonic development.
        Reprod Fertil Dev. 2008; 20: 875-883
        • Coward K.
        • Ponting C.P.
        • Chang H.Y.
        • Hibbitt O.
        • Savolainen P.
        • Jones K.T.
        • et al.
        Phospholipase Czeta, the trigger of egg activation in mammals, is present in a nonmammalian species.
        Reproduction. 2005; 130: 157-163
        • Ito M.
        • Shikano T.
        • Oda S.
        • Horiguchi T.
        • Tanimoto S.
        • Awaji T.
        • et al.
        Difference in Ca2+ oscillation–inducing activity and nuclear translocation ability of PLCZ1, an egg-activating sperm factor candidate, between mouse, rat, human, and medaka fish.
        Biol Reprod. 2008; 78: 1081-1090
        • Coward K.
        • Ponting C.P.
        • Zhang N.
        • Young C.
        • Huang C.J.
        • Chou C.M.
        • et al.
        Identification and functional analysis of an ovarian form of the egg activation factor phospholipase C zeta (PLCζ) in pufferfish.
        Mol Reprod Dev. 2011; 78: 48-56
        • Mizushima S.
        • Takagi S.
        • Ono T.
        • Atsumi Y.
        • Tsukada A.
        • Saito N.
        • et al.
        Developmental enhancement of intracytoplasmic sperm injection (ICSI)–generated quail embryos by phospholipase C zeta cRNA.
        J Poultry Sci. 2008; 45: 152-158
        • Yoon S.Y.
        • Fissore R.A.
        Release of phospholipase C zeta and [Ca2+]i oscillation–inducing activity during mammalian fertilization.
        Reproduction. 2007; 134: 695-704
        • Young C.
        • Grasa P.
        • Coward K.
        • Davis L.C.
        • Parrington J.
        Phospholipase C zeta undergoes dynamic changes in its pattern of localization in sperm during capacitation and the acrosome reaction.
        Fertil Steril. 2009; 91: 2230-2242
        • Grasa P.
        • Coward K.
        • Young C.
        • Parrington J.
        The pattern of localization of the putative oocyte activation factor, phospholipase C zeta, in uncapacitated, capacitated, and ionophore-treated human spermatozoa.
        Hum Reprod. 2008; 23: 2513-2522
        • Nakai M.
        • Ito J.
        • Sato K.I.
        • Noguchi J.
        • Kaneko H.
        • Kashiwazaki N.
        • et al.
        Pre-treatment of sperm reduces success of intracytoplasmic sperm injection in the pig.
        Reproduction. 2011; 142: 285-293
        • Heindryckx B.
        • van der Elst J.
        • De Sutter P.
        • Dhont M.
        Treatment option for sperm- or oocyte-related fertilization failure: assisted oocyte activation following diagnostic heterologous ICSI.
        Hum Reprod. 2005; 20: 2237-2241
        • Yanagida K.
        • Fujikura Y.
        • Katayose H.
        The present status of artificial oocyte activation in assisted reproductive technology.
        Reprod Med and Biology. 2008; 7: 133-142
        • 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: 662-668
        • Yoon S.Y.
        • Jellerette T.
        • Salicioni A.M.
        • Lee H.C.
        • Yoo M.S.
        • Coward K.
        • et al.
        Human sperm devoid of PLC, zeta 1 fail to induce Ca2+ release and are unable to initiate the first step of embryo development.
        J Clin Invest. 2008; 118: 3671-3681
        • Taylor S.L.
        • Yoon S.Y.
        • Morshedi M.S.
        • Lacey D.R.
        • Jellerette T.
        • Fissore R.A.
        • et al.
        Complete globozoospermia associated with PLCzeta deficiency treated with calcium ionophore and ICSI results in pregnancy.
        Reprod Biomed Online. 2010; 20: 559-564
        • Kashir J.
        • Jones C.
        • Lee H.C.
        • Rietdorf K.
        • Nikiforaki D.
        • Durrans C.
        • et al.
        Loss of activity mutations in phospholipase C zeta (PLCζ) abolishes calcium oscillatory ability of human recombinant protein in mouse oocytes.
        Hum Reprod. 2011; 26: 3372-3387
        • Heytens E.
        • Parrington J.
        • Coward K.
        • Young C.
        • Lambrecht S.
        • Yoon S.Y.
        • et al.
        Reduced amounts and abnormal forms of phospholipase C zeta in spermatozoa from infertile men.
        Hum Reprod. 2009; 24: 2417-2428
        • Nomikos M.
        • Elgmati K.
        • Theodoridou M.
        • Calver B.L.
        • Cumbes B.
        • Nounesis G.
        • et al.
        Male infertility-linked point mutation disrupts the Ca2+ oscillation–inducing and PIP2 hydrolysis activity of sperm PLCζ.
        Biochem J. 2011; 434: 211-217
        • Kashir J.
        • Konstantinidis M.
        • Jones C.
        • Lemmon B.
        • Lee H.C.
        • Hamer R.
        • et al.
        A maternally inherited autosomal point mutation in human phospholipase C zeta (PLCζ) leads to male infertility.
        Hum Reprod. 2012; 27: 222-231
        • 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: 263-267
        • Bi Y.
        • Xu W.M.
        • Wong H.Y.
        • Zhu H.
        • Zhou Z.M.
        • Chan H.C.
        • et al.
        NYD-SP27, a novel intrinsic decapacitation factor in sperm.
        Asian J Androl. 2009; 11: 229-239
        • Allamaneni S.S.
        • Agarwal A.
        • Rama S.
        • Ranganathan P.
        • Sharma R.K.
        Comparative study on density gradients and swim-up preparation techniques utilizing neat and cryopreserved spermatozoa.
        Asian J Androl. 2005; 7: 86-92
        • Yu Y.
        • Saunders C.M.
        • Lai F.A.
        • Swann K.
        Preimplantation development of mouse oocytes activated by different levels of human phospholipase C zeta.
        Hum Reprod. 2008; 23: 365-373
        • Cooney M.A.
        • Malcuit C.
        • Cheon B.
        • Holland M.K.
        • Fissore R.A.
        • d'Cruz N.T.
        Species-specific differences in the activity and nuclear localization of murine and bovine phospholipase C zeta 1.
        Biol Reprod. 2010; 83: 92-101
        • Kaewmala K.
        • Uddin M.
        • Cinar M.
        • Große-Brinkhaus C.
        • Jonas E.
        • Tesfaye D.
        • et al.
        Investigation into association and expression of PLCz and COX-2 as candidate genes for boar sperm quality and fertility.
        Reprod Domest Anim. 2011; 47: 213-223
        • Aghajanpour S.
        • Ghaedi K.
        • Salamian A.
        • Deemeh M.R.
        • Tavalaee M.
        • Moshtaghian J.
        • et al.
        Quantitative expression of phospholipase C zeta, as an index to assess fertilization potential of a semen sample.
        Hum Reprod. 2011; 26: 2950-2956
        • Platts A.E.
        • Dix D.J.
        • Chemes H.E.
        • Thompson K.E.
        • Goodrich R.
        • Rockett J.C.
        • et al.
        Success and failure in human spermatogenesis as revealed by teratozoospermic RNAs.
        Hum Mol Genet. 2007; 16: 763-773
        • Lalancette C.
        • Miller D.
        • Li Y.
        • Krawetz S.A.
        Paternal contributions: new functional insights for spermatozoal RNA.
        J Cell Biochem. 2008; 104: 1570-1579
        • Bettegowda A.
        • Wilkinson M.F.
        Transcription and post-transcriptional regulation of spermatogenesis.
        Philos Trans R Soc Lond B Biol Sci. 2010; 365: 1637-1651
        • Ogonuki N.
        • Inoue K.
        • Ogura A.
        Birth of normal mice following round spermatid injection without artificial oocyte activation.
        J Reprod Dev. 2011; 57: 534-538
        • Yazawa H.
        • Yanagida K.
        • Sato A.
        Human round spermatids from azoospermic men exhibit oocyte-activation and Ca2+ oscillation–inducing activities.
        Zygote. 2007; 15: 337-346
        • Yoneda A.
        • Kashima M.
        • Yoshida S.
        • Terada K.
        • Nakagawa S.
        • Sakamoto A.
        • et al.
        Molecular cloning, testicular postnatal expression, and oocyte-activating potential of porcine phospholipase C zeta.
        Reproduction. 2006; 132: 393-401
        • Ishikawa T.
        • Shiotani M.
        • Izumi Y.
        • Hashimoto H.
        • Kokeguchi S.
        • Goto S.
        • et al.
        Fertilization and pregnancy using cryopreserved testicular sperm for intracytoplasmic sperm injection with azoospermia.
        Fertil Steril. 2009; 92: 174-179
        • Yazawa H.
        • Yanagida K.
        • Katayose H.
        • Hayashi S.
        • Sato A.
        Comparison of oocyte activation and Ca2+ oscillation–inducing abilities of round/elongated spermatids of mouse, hamster, rat, rabbit and human assessed by mouse oocyte activation assay.
        Hum Reprod. 2000; 15: 2582-2590
        • Larsen L.
        • Scheike T.
        • Jensen T.K.
        • Bonde J.P.
        • Ernst E.
        • Hjollund N.H.
        • et al.
        • Danish First Pregnancy Planner Study Team
        Computer-assisted semen analysis parameters as predictors for fertility of men from the general population.
        Hum Reprod. 2000; 15: 1562-1567
        • Wu B.
        • Wong D.
        • Lu S.
        • Dickstein S.
        • Silva M.
        • Gelety T.J.
        Optimal use of fresh and frozen-thawed testicular sperm for intracytoplasmic sperm injection in azoospermic patients.
        J Assist Reprod Genet. 2005; 22: 389-394
      1. Pacey AA. Assessment of male factor. Best Pract Res Clin Obstet Gynaecol. Published online 2012.

        • Stalf T.
        • Mehnert C.
        • Hajimohammad A.
        • Manolopoulos K.
        • Shen Y.
        • Schuppe H.C.
        • et al.
        Influence of motility and vitality in intracytoplasmic sperm injection with ejaculated and testicular sperm.
        Andrologia. 2005; 37: 125-130