Phospholipase C zeta (PLC?): Oocyte activation and clinical links to male factor infertility. Amdani SN 2013 et al.
Mounting scientific and clinical evidence supports the key role played by phospholipase C zeta (PLC?), a sperm-specific protein, in the activation of oocytes following fertilisation. Lacking a pleckstrin homology domain, PLC? remains the smallest known mammalian PLC and was first identified in 2002. Since then, PLC? has been the target for a multitude of studies in both mammalian and non-mammalian species focused upon its fundamental biochemical activity and crucial role as the mediator of oocyte activation. The earliest event subsequent to gamete fusion is the onset of a series of intracellular calcium oscillations within the oocyte, which are known to modulate cortical granule exocytosis, release meiotic arrest, regulate gene expression, recruit maternal mRNA, and initiate embryogenesis. Collectively these processes are known as 'oocyte activation' and together, represent a fundamental mechanism for early embryonic development. Evidence suggests that these processes are initiated and controlled by calcium release from ooplasmic sources in response to PLC? activity via the inositol-1,4,5-triphosphate (IP3) pathway. While the biochemical action of PLC? has been extensively studied, especially in relation to the EF-hands, X-Y linker, and C2 domain, all of which play critical roles for invivo activity, there are still key gaps in our knowledge, particularly in terms of regulation and interaction with other proteins within the oocyte. Moreover, increasing clinical evidence has revealed a strong correlation between certain types of male infertility and the aberrant expression, localisation, structure and function of PLC? in human sperm, particularly in cases of recurrent intracytoplasmic sperm injection (ICSI) failure, globozoospermia, and oocyte activation deficiency (OAD). In addition, two heterozygous substitution mutations have been identified in the coding sequence of PLC? in one particular patient causing disruption to the catalytic X and Y domains and resulting in infertility. Although, such cases can be treated via the use of artificial oocyte activators (AOAs) such as calcium ionophores, significant concern remains over the use of such chemical agents, largely due to the fact that calcium release manifests as a single transient, rather than a series of oscillations as observed during normal fertilisation. Current interest in PLC? is thus to develop a series of prognostic, diagnostic and therapeutic approaches which could first identify male patients that are deficient in PLC? and then rescue oocyte activation ability via assisted reproductive technology (ART) and a pure, functionally-active, recombinant human PLC? protein. While significant progress has been made in such areas over recent years, there is a clear need to translate scientific findings to clinical settings in order to maximise successful outcome for patients.
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Rogers NT et al reported that Phospholipase C{zeta} causes Ca2+ oscillations and parthenogenetic activation of human oocytes.
At fertilization in mammals the sperm activates development of the oocyte by inducing a prolonged series of oscillations in the cytosolic free Ca(2+) concentration. One theory of signal transduction at fertilization suggests that the sperm cause the Ca(2+) oscillations by introducing a protein factor into the oocyte after gamete membrane fusion. We recently identified this sperm-specific protein as phospholipase Czeta (PLCzeta), and we showed that PLCzeta triggers Ca(2+) oscillations in unfertilized mouse oocytes. Here we report that microinjection of the complementary RNA for human PLCzeta causes prolonged Ca(2+) oscillations in aged human oocytes that had failed to fertilize during in vitro fertilization or intracytoplasmic sperm injection. The frequency of Ca(2+) oscillations was related to the concentration of complementary RNA injected. At low concentrations, PLCzeta stimulated parthenogenetic activation of oocytes. These embryos underwent cleavage divisions and some formed blastocysts. These data show that PLCzeta is a novel parthenogenetic stimulus for human oocytes and that it is unique in its ability to mimic the repetitive nature of the Ca(2+) stimulus provided by the sperm during human fertilization.
NCBI Summary:
The protein encoded by this gene is a member of the phosphoinositide-specific phospholipase C family. Members in this family, classified into six isotypes that are tissue- and organ-specific, hydrolyze phosphatidylinositol 4,5-bisphosphate just before the phosphate group to yield diacylglycerol and inositol 1,4,5-trisphosphate. This protein localizes to the acrosome in spermatozoa and elicits Ca(2+) oscillations and egg activation during fertilization that leads to early embryonic development. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Aug 2012]
General function
Enzyme
Comment
Species-Specific Differences in the Activity and Nuclear Localization of Murine and Bovine Phospholipase C, Zeta 1. Cooney MA et al. Injection of mammalian sperm extracts or cRNA of the sperm-specific phospholipase C, zeta 1 (PLCZ1) have been shown to trigger repetitive oscillations in the concentration of free calcium ([Ca(2+)](i)), leading to oocyte activation and embryo development in all mammals studied to date. While PLCZ1 has cross-species activity, it has also been observed that species-specific differences may exist in the frequency and pattern of the resulting [Ca(2+)](i) oscillations following PLCZ1 cRNA injection into oocytes of different species. Accordingly, we used a cross-over design strategy to directly investigate the activity of murine and bovine PLCZ1 in both murine and bovine oocytes. In murine oocytes, injection of murine Plcz1 cRNA induced [Ca(2+)](i) oscillations at ten-fold lower concentrations than bovine PLCZ1, although in bovine oocytes, bovine PLCZ1 was more effective than murine Plcz1 at inducing [Ca(2+)](i) oscillations. Investigation of IPTR1 (IP(3)R1) down-regulation in bovine oocytes by PLCZ1 cRNA also showed that bovine PLCZ1 was more active in homologous oocytes. To determine whether these PLCZs exhibited similar cellular distribution, Venus-tagged PLCZ1 cRNA was injected into oocytes and PLCZ1 overexpressed. Bovine PLCZ1 failed to accumulate in the pronuclei (PN) of bovine or murine zygotes despite possessing a putative nuclear localization signal. Conversely, murine PLCZ1 accumulated in the PN of both murine and bovine zygotes. These results demonstrate that murine and bovine PLCZ1 possess species-specific differences in activity and suggest potential differences in the mode of action of the protein between the two species. Variation in sperm PLCZ1 protein content among species, along with oocyte-specific differences in the localization and availability of PLCZ1 substrates, may further contribute to optimize the activation stimulus to enhance embryo development.
Cellular localization
Cytoplasmic
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Ovarian function
Early embryo development
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Phospholipase C-zeta deficiency as a cause for repetitive oocyte fertilization failure during ovarian stimulation for in vitro fertilization with ICSI: a case report. Chithiwala ZH et al. (2015) The purpose of this study is to describe impaired oocyte fertilization from phospholipase C-zeta (PLC-ζ) deficiency in normal-appearing sperm that was successfully treated using calcium (Ca(2+)) ionophore with intracytoplasmic sperm injection (ICSI) of oocytes matured in vitro. An infertile couple undergoing in vitro fertilization (IVF) experienced failed oocyte fertilization following ICSI with normal-appearing sperm. A semen sample collected from the patient was used to assess the expression of sperm PLC- ζ protein by Western blot analysis and immunofluorescence and PLC-ζ bioactivity by an in vitro model of Ca(2+) release. A second IVF cycle was performed using Ca(2+) ionophore with ICSI to enhance Ca(2+)-induced oocyte activation of oocytes matured in vitro. Sperm PLC-ζ protein deficiency was demonstrated by Western blot analysis and immunofluorescence and confirmed by reduced PLC-ζ bioactivity using an in vitro model of Ca(2+) release. Nevertheless, with this sperm and supplementation of Ca(2+) ionophore following ICSI, fertilization of four of six oocytes matured in vitro was obtained. In addition, four embryos underwent cleavage and two of them reached the blastocyst stage. Transfer of these blastocysts into the uterus led to a single pregnancy and live birth. Deficiency of PLC-ζ in normal-appearing human sperm is associated with impaired Ca(2+)-dependent oocyte activation during ICSI. Under this condition, use of Ca(2+) ionophore following ICSI of oocytes matured in vitro improves embryo developmental competence, possibly through the activation of Ca(2+)-dependent mechanisms governing fertilization and preimplantation embryogenesis.//////////////////
Microinjection of mouse phospholipase Czeta complementary RNA into mare oocytes induces long-lasting intracellular calcium oscillations and embryonic development. Bedford-Guaus SJ et al. Methods presently used to activate mare oocytes for assisted reproduction technologies provide low rates of advanced embryonic development. Because phospholipase Czeta (PLCzeta) is the postulated sperm-borne factor responsible for oocyte activation at fertilisation, the aim of the present study was to investigate the pattern of [Ca(2+)](i) oscillations and developmental rates achieved by microinjection of three concentrations of mouse PLCzeta complementary (c) RNA (1, 0.5 or 0.25 mug muL(-1)) into mare oocytes. The frequency of [Ca(2+)](i) oscillations was no different (P > 0.05) after injection of 1, 0.5 or 0.25 mug muL(-1) PLCzeta cRNA (41.1 +/- 5.3, 47 +/- 4.0 and 55.4 +/- 9.0, respectively). However, [Ca(2+)](i) oscillations persisted longest (P < 0.05) for oocytes injected with 0.5 mug muL(-1) PLCzeta cRNA (570.7 +/- 64.2 min). There was no significant difference in cleavage rates after injection of the three concentrations of PLCzeta (P > 0.05; range 97-100%), but the proportion of oocytes reaching advanced stages of embryonic development (>64 nuclei) was significantly lower for oocytes injected with 0.25 mug muL(-1) PLCzeta cRNA (3%) than for those injected with 1 mug muL(-1) PLCzeta cRNA (15%). Based on these results, microinjection of PLCzeta may prove an effective and consistent method for the parthenogenetic activation of mare oocytes for nuclear transfer and provides a physiologically relevant tool with which to study fertilisation-dependent [Ca(2+)](i) signalling in this species.
Expression regulated by
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Ovarian localization
sperm
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Follicle stages
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Phenotypes
Mutations
2 mutations
Species: human
Mutation name: type: naturally occurring fertility: infertile - non-ovarian defect Comment: Novel phospholipase C zeta 1 mutations associated with fertilization failures after ICSI. Torra-Massana M et al. (2019) Are phospholipase C zeta 1 (PLCZ1) mutations associated with fertilization failure (FF) after ICSI? New mutations in the PLCZ1 sequence are associated with FFs after ICSI. FF occurs in 1-3% of ICSI cycles, mainly due to oocyte activation failure (OAF). The sperm PLCζ/PLCZ1 protein hydrolyzes phosphatidylinositol (4, 5)-bisphosphate in the oocyte, leading to intracellular calcium release and oocyte activation. To date, few PLCZ1 point mutations causing decreased protein levels or activity have been linked to FF. However, functional alterations of PLCζ/PLCZ1 in response to both described and novel mutations have not been investigated. We performed a study including 37 patients presenting total or partial FF (fertilization rate (FR), ≤25%) after ICSI occurring between 2014 and 2018. Patients were divided into two groups based on oocyte evaluation 19 h post ICSI: FF due to a defect in oocyte activation (OAF, n = 22) and FF due to other causes ('no-OAF', n = 15). Samples from 13 men with good fertilization (FR, >50%) were used as controls. PLCζ/PLCZ1 protein localization and levels in sperm were evaluated by immunofluorescence and western blot, respectively. Sanger sequencing on genomic DNA was used to identify PLCZ1 mutations in exonic regions. The effect of the mutations on protein functionality was predicted in silico using the MODICT algorithm. Functional assays were performed by cRNA injection of wild-type and mutated forms of PLCZ1 into human in vitro matured metaphase II oocytes, and fertilization outcomes (second polar body extrusion, pronucleus appearance) scored 19 h after injection. In the OAF group, 12 (54.6%) patients carried at least one mutation in the PLCZ1 coding sequence, one patient out of 15 (6.7%) in the no-OAF group (P < 0.05) and none of the 13 controls (P < 0.05). A total of six different mutations were identified. Five of them were single-nucleotide missense mutations: p.I120M, located at the end of the EF-hand domain; p.R197H, p.L224P and p.H233L, located at the X catalytic domain; and p.S500 L, located at the C2 domain. The sixth mutation, a frameshift variant (p.V326K fs*25), generates a truncated protein at the X-Y linker region. In silico analysis with MODICT predicted all the mutations except p.I120M to be potentially deleterious for PLCζ/PLCZ1 activity. After PLCZ1 cRNA injection, a significant decrease in the percentage of activated oocytes was observed for three mutations (p.R197H, p.H233L and p.V326K fs*25), indicating a deleterious effect on enzymatic activity. PLCZ1 protein localization and expression levels in sperm were similar across groups. FRs were restored (to >60%) in patients carrying PLCZ1 mutations (n = 10) after assisted oocyte activation (AOA), with seven patients achieving pregnancy and live birth. Caution should be exerted when comparing the cRNA injection results with fertilization outcomes after ICSI, especially in patients presenting mutations in heterozygosis. PLCZ1 mutations were found in high frequency in patients presenting OAF. Functional analysis of three mutations in human oocytes confirms alteration of PLCζ/PLCZ1 activity and their likely involvement in impaired oocyte activation. Our results suggest that PLCZ1 gene sequencing could be useful as a tool for the diagnosis and counseling of couples presenting FF after ICSI due to OAF. This work was supported by intramural funding of Clínica EUGIN, by the Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia (GENCAT 2015 DI 049 to M. T.-M. and GENCAT 2015 DI 048 to D. C.-B.) and by the Torres Quevedo Program from the Spanish Ministry of Economy and Competitiveness to A. F.-V. No competing interest declared.//////////////////
Species: human
Mutation name: type: naturally occurring fertility: infertile - non-ovarian defect Comment: The identification of novel mutations in PLCZ1 responsible for human fertilization failure and a therapeutic intervention by artificial oocyte activation. Mu J et al. (2020) Fertilization involves a series of molecular events immediately following egg-sperm fusion; Ca2+ oscillations are the earliest signaling event and they initiate the downstream reactions including pronucleus formation. Successful human reproduction requires normal fertilization. In clinical IVF or ICSI attempts, some infertile couples suffer from recurrent fertilization failure. However, the genetic reasons for fertilization failure are largely unknown. Here, we recruited several couples diagnosed with fertilization failure even though their gametes are morphologically normal. Through whole-exome sequencing and Sanger sequencing, we identified biallelic mutations in gene encoding phospholipase C zeta 1 (PLCZ1) in four independent males in couples diagnosed with fertilization failure. Western blotting showed that missense mutations decreased the level of PLCZ1 and that nonsense or frameshift mutations resulted in undetectable or truncated proteins. Expression of these mutations in mice significantly reduced the levels of oocyte activation. Artificial oocyte activation in patient oocytes could rescue the phenotype of fertilization failure and help establish pregnancy and lead to live birth. Our findings expand the spectrum of PLCZ1 mutations that are responsible for human fertilization failure and provide a potentially feasible therapeutic treatment for these patients.//////////////////