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PLC{zeta}: a sperm-specific trigger of Ca2+ oscillations in eggs and embryo development

Christopher M. Saunders1, Mark G. Larman2, John Parrington3, Llewellyn J. Cox1, Jillian Royse1, Lynda M. Blayney1, Karl Swann2 and F. Anthony Lai1,*

1 Cell Signalling Laboratory, Wales Heart Research Institute, University of Wales College of Medicine, Cardiff CF14 4XN, UK
2 Department of Anatomy and Developmental Biology, University College, London WC1E 6BT, UK
3 Department of Physiology, University College, London WC1E 6BT, UK



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  		Fig. 1. Identification of a novel sperm PLC. (A) Immunoblot analysis of heparin-eluted soluble extracts of brain, kidney, liver and sperm (lanes B, K, L, S; 50 µg/lane) from mouse, boar and hamster, using antibody raised to the novel sperm PLC. Molecular weight markers in kDa, on the right. (B) Immunoblot of heparin-eluted soluble sperm proteins fractionated by gel filtration column chromatography on Sephacryl S-200 column. The underlined 150 kDa and 29 kDa indicate elution positions of gel filtration standards alcohol dehydrogenase and carbonic anhydrase, respectively. Shown below is the corresponding Ca2+ release activity of column fractions B, D and F assayed fluorometrically in sea urchin egg homogenates. Scale bars indicate time (seconds) and relative fluorescence units (RFU) (Jones et al., 1998bGo). Arrows indicate time of addition.

 


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  		Fig. 2. Molecular cloning of mouse sperm PLC{zeta}. (A) Clustal alignment of mouse sperm PLC{zeta} with rat PLC{delta}1 (Accession number, P10688). Identical amino acids are shown in shaded black boxes, conservative substitutions in grey. (B) Schematic illustrating the predicted domain features of mouse PLC{zeta} and mammalian PLC isoforms ß, {gamma}, {delta}, and {epsilon}. (C) Sequence identity (blue) and similarity (red) between mammalian PLC isoforms (ß3, P51432; {gamma}2, AAH07565; {delta}1, P10688; {epsilon}, AAG17145; and {zeta}). (D) Dendrogram illustrating phylogeny of Clustal aligned mammalian PLC sequences. Tree branch lengths, indicating amino acid substitutions per residue, were 0.298 for {zeta}; 0.309-0.322 for {delta}1-4; 0.397 for {epsilon}; 0.400-0.413 for ß1-4; and 0.412-0.417 for {gamma}1-2.

 


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  		Fig. 3. Sperm-specific expression of mouse PLC{zeta}. (A) Northern blot analysis of PLC{zeta} transcript distribution in mouse. Lanes from left to right: RNA standard markers, brain, heart, kidney, liver, lung, skeletal muscle, spleen, testis (2 µg polyA+-RNA/lane). Molecular weight markers in kb are on the left. (B) Immunoblot analysis of PLC{zeta} protein distribution in mouse. Left to right: brain, heart, kidney, liver, lung, skeletal muscle, sperm (50 µg protein/lane). Molecular weight markers in kDa are on the right. (C) Polymerase chain reaction detection of PLC{zeta} in cDNA from mouse spermatid and mouse testis devoid of spermatids. Left to right: DNA markers (2.0, 1.6, 1.0, 0.8, 0.6 and 0.5 kb, top to bottom), spermatid cDNA (10 ng), testis cDNA (10 ng), blank (no DNA) and positive control (1 ng PLC{zeta} plasmid).

 


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  		Fig. 4. PLC{zeta} triggers Ca2+ oscillations in MII-arrested mouse eggs. (A) Dose-dependent Ca2+ oscillations in fura-red loaded mouse eggs triggered by microinjection of cRNA encoding mouse sperm PLC{zeta} (2 and 0.002 mg/ml, top and middle trace, respectively) and after preincubation with 10 µM cycloheximide (0.02 mg/ml, bottom trace). (B) Mean interspike interval of Ca2+ oscillations in eggs following microinjection of various PLC{zeta} cRNA concentrations (2-0.002 mg/ml in pipette, i.e. <0.1-0.0001 mg/ml in egg) compared with the interval observed upon in vitro fertilisation (IVF). Number of microinjected eggs is shown above each condition. *, significantly different from IVF at the 5% level (Student’s unpaired t-test).

 


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  		Fig. 5. In vitro fertilisation consistent with PLC{zeta}-induced Ca2+ oscillations. Ca2+ changes in fura-red loaded mouse eggs that were either (A) in vitro fertilised with mouse sperm, or microinjected with cRNA encoding (B) PLC{zeta} at 0.02 mg/ml; (C) PLC{delta}1 at 2 mg/ml; (D) {Delta}PHPLC{delta}1 at 2 mg/ml (PH domain-deleted PLC{delta}1); (E) D210RPLC{zeta} at 2 mg/ml. (A-I,B-I) Expanded traces of the longer-duration, first Ca2+ transient taken from A,B, respectively. (F) Autoradiograph following SDS-PAGE of [35S]-labelled protein expressed in vitro from cRNA, lanes from left to right, of D210RPLC{zeta}, PLC{zeta}, {Delta}PHPLC {delta}1 and PLC{delta}1 corresponding to predicted protein sizes of 74, 74, 70 and 85 kDa, respectively.

 


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  		Fig. 6. PLC{zeta} quantitation in cRNA-microinjected eggs and mouse sperm. (A) Ca2+ changes in a fura-red loaded mouse egg microinjected with cRNA encoding Myc-PLC{zeta} at 0.02 mg/ml. (B) Immunoblot analysis of Myc immunoreactive protein in uninjected (U) and Myc-PLC{zeta} cRNA-injected (I) mouse eggs (240 eggs/lane, 2 mg/ml, 5 hours post-injection). Molecular weight markers in kDa are on right. (C) Immunoblot and relative mobility analysis of native PLC{zeta} in mouse sperm (Sp, left panel, anti-PLC{zeta} antibody) and of Myc-PLC{zeta} in mouse eggs (right panel, anti-Myc antibody) microinjected with 1.0, 0.3 and 0.1 mg/ml Myc-PLC{zeta} cRNA (100 eggs/lane, 5 hours post-injection). 80 kDa protein marker is on the left. (D) Densitometric calibration plot of E. coli-purified Myc-PLC{zeta}-Histag protein using anti-PLC{zeta} antibody. Correlation coefficient, r=0.99. Broken line indicates interpolation of PLC{zeta} protein content corresponding to sperm extract derived from 4x105 mouse sperm.

 


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  		Fig. 7. Ca2+ release activity in PLC{zeta}-immunodepleted soluble sperm extracts. (A) Immunoblot analysis of PLC{zeta} protein in hamster sperm extract supernatants after incubation with control IgG or anti-PLC{zeta} antibody (S- and S+, respectively) and the corresponding precipitated proteins bound to control IgG beads or anti-PLC{zeta} beads (P- and P+, respectively). (B) Ca2+ release activity of antibody-treated sperm supernatants, S– and S+, assayed fluorometrically in sea urchin egg homogenates. Scale bars indicate time (seconds) and relative fluorescence units (RFU). Arrows indicate time of addition (C) Ca2+ changes in fura-red loaded mouse eggs after microinjection with sperm extract that was either untreated (top trace), control IgG-treated (second trace, n=13) or anti-PLC{zeta} antibody-treated (bottom two traces, n=13). In 6/13 cases (third trace), the anti-PLC{zeta} antibody-treated sperm extract showed an injection artifact-related single Ca2+ spike; in other cases there was no Ca2+ change (fourth trace).

 


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  		Fig. 8. Activation and embryo development to blastocyst in PLC{zeta}-injected mouse eggs. (A) Mouse eggs were either microinjected with PLC{zeta} cRNA (0.02 mg/ml), or parthenogenetically activated with strontium (5 mM, 4 hours) or fertilised with sperm in vivo, then placed in a 5% CO2 incubator at 37°C. Percentage of eggs reaching the two-cell stage after 24 hours, and morula/blastocyst stage after 96 hours, was recorded for each treatment. Number of microinjected eggs is shown above each condition. (B) Micrographs illustrating mouse embryos at the two-cell stage (left) and blastocyst stage (right), at 24 hours and 96 hours, respectively, after egg microinjection with PLC{zeta} cRNA (0.02 mg/ml). (C) Micrograph illustrating mouse egg 24 hours after microinjection with D210RPLC{zeta} cRNA (0.02 mg/ml).

 




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