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: a sperm-specific trigger of Ca2+ oscillations in eggs and embryo development
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
*Author for correspondence (e-mail: lait{at}cf.ac.uk)
Accepted 1 May 2002
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, that triggers Ca2+ oscillations in mouse eggs indistinguishable from those at fertilisation. PLC removal from sperm extracts abolishes Ca2+ release in eggs. Moreover, the PLC content of a single sperm was sufficient to produce Ca2+ oscillations as well as normal embryo development to blastocyst. Our results are consistent with sperm PLC as the molecular trigger for development of a fertilised egg into an embryo.
Key words: Fertilisation, Sperm factor, Phospholipase C, Ca2+ oscillations, Egg activation, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In mammals, the unification of sperm and egg leads to a distinctive series of cytosolic Ca2+ oscillations that are a prerequisite for normal embryo development (Miyazaki et al., 1993; Stricker, 1999). This striking Ca2+ signalling phenomenon arises from increases in inositol 1,4,5-trisphosphate (IP3) levels, which activate IP3 receptor-mediated Ca2+ release from intracellular stores in the egg (Miyazaki et al., 1993; Brind et al., 2000; Jellerette et al., 2000). However, the basic mechanism that results in stimulation of phosphoinositide (PI) metabolism following sperm-egg interaction has not been determined in any species.
The sperm factor hypothesis of signalling at fertilisation proposes that spermatozoa contain a soluble Ca2+ releasing factor that enters the egg after the gamete membranes fuse together and generates Ca2+ oscillations (Swann, 1990; Stricker, 1999). This is consistent with the finding that cytoplasmic fusion of sperm and egg is a prelude to Ca2+ release (Lawrence et al., 1997; Jones et al., 1998a). Direct support for this hypothesis comes from experiments where microinjection into eggs of either single spermatozoa, or soluble sperm extracts, triggers Ca2+ oscillations similar to those at fertilisation in mammalian and some non-mammalian eggs (Swann, 1990; Wu et al., 1997; Wu et al., 1998; Stricker, 1997; Nakano et al., 1997; Kyozuka et al., 1998; Tang et al., 2000). The mammalian sperm factor that generates Ca2+ oscillations is protein based (Swann, 1990), acts across species (Wu et al., 1997), and can cause Ca2+ release in somatic cells (Berrie et al., 1996) and in cell-free systems such as sea urchin egg homogenates (Jones et al., 1998b). Sperm specifically express a Ca2+ oscillation-inducing protein, because microinjecting mRNA isolated from spermatogenic cells, but not mRNA from other tissues, elicits fertilisation-like Ca2+ oscillations in mouse eggs (Parrington et al., 2000). Despite intensive biochemical investigation, the molecular identity of the putative sperm factor has remained elusive (Stricker, 1999). Different proteins, including a 33 kDa protein (Parrington et al., 1996) and a truncated form of the Kit receptor (Sette et al., 1997), have previously been sperm factor candidates. However, neither these two, nor any other sperm proteins, have been shown to generate Ca2+ oscillations in eggs (Wu et al., 1998; Wolosker et al., 1998), the single-most distinctive feature of mammalian fertilisation (Stricker, 1999).
In intact eggs and egg homogenates, mammalian sperm extracts trigger Ca2+ release by stimulating IP3 production (Jones et al., 1998b; Rice et al., 2000; Jones et al., 2000; Wu et al., 2001), indicating involvement of a PI-specific phospholipase C (PLC) in the signal transduction mechanism. The high level of PLC enzyme activity measured biochemically in sperm extracts suggests that the sperm factor may itself be a PLC (Jones et al., 1998b; Rice et al., 2000). However, the PLCß, 
and 
isoforms that exist in sperm are absent from chromatographic fractions of sperm extract that specifically cause Ca2+ oscillations (Wu et al., 2001; Parrington et al., 2002). In addition, when the purified, recombinant PLCß1, 1, 2 or 1 proteins are added to egg homogenates or microinjected into eggs, they fail to cause Ca2+ release (Jones et al., 2000). A PLC4 splice variant found in sperm functions in the acrosome reaction, rather than in Ca2+ release in eggs at fertilisation (Fukami et al., 2001). These observations led us to investigate the possible existence of a distinct, uncharacterised sperm PLC isoform. Our studies reveal that a new PLC isoform (PLC), specifically expressed in mammalian sperm, uniquely possesses all the essential properties of the sperm factor. These results are consistent with sperm PLC as the physiological trigger of egg activation, and thus an essential protein for mammalian fertilisation and embryo development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), were separated by 10% SDS-PAGE, transferred to PVDF membrane and probed with anti-peptide antisera. Heparin affinity and gel filtration column chromatography of soluble sperm proteins (10 mg) used an AKTA FPLC system (Amersham Pharmacia) (Parrington et al., 1996) in 50 mM sodium phosphate, 0.15 M NaCl, pH 7. Fluorometric Ca2+ release assays using fluo-3 in sea urchin homogenates (Jones et al., 1998b) was monitored using an LS50B (Perkin-Elmer).
Molecular cloning and sequence analysis of mouse sperm PLC
Blast searches of the mouse EST database using mammalian PLC sequences (www.ncbi.nlm.nih.gov/BLAST) identified 12, novel PLC-related sequences (Accession Numbers, AV282878, AV278700, AV278207, AV272100, AV271735, AV270614, AV270212, AV263382, AV263095, AV258739, AV258594 and AV045146). These mouse ESTs were 232-294 basepairs with identical 3' sequences and all derived from testis. The full-length sequence encoding this novel PLC, named PLC, was obtained by two-step RACE PCR amplification with pfu polymerase from a mouse spermatid cDNA library (35 ng) in lambdaZAPII. The single amplified DNA of 2.2 kb was cloned into pCR-XL-TOPO (Invitrogen), ten independent colonies were sequenced on both strands, and analysed for open reading frame by MacVector 6.5 (Oxford Molecular), for PLC homology and phylogeny by ClustalW sequence alignment (www.clustalw.genome.ad.jp) and domain structure by RPS-Blast (www.ncbi.nlm.nih.gov/structure/cdd). The GenBank Accession Number for PLC is AF435950.
Northern blot and polymerase chain reaction analysis
A 1.2 kb probe from the 5' end of mouse PLC, prepared by PCR as above, was cloned into pCR-BluntII-TOPO (Invitrogen) and sequenced. Antisense digoxigenin-labelled RNA synthesised from this plasmid (DIG Nucleic acid labelling system, Roche Molecular Biochemicals) was used to probe a male mouse tissue polyA+-RNA blot with equal loading of 2 µg polyA+-RNA/lane (MessageMap Northern, Stratagene). Hybridised probe was detected using the DIG Luminescence Detection Kit (Roche Molecular Biochemicals) and displayed using QuantityOne software (BioRad). Polymerase chain reaction amplification using 30 cycles was performed with oligonucleotide primers that define a 0.9 kb region within PLC, using cDNA prepared from mouse spermatids or mouse testis devoid of spermatids in the lambda ZAPII vector (10 ng). Negative and positive controls comprised reactions without DNA template and with PLC plasmid DNA (1 ng), respectively.
Complementary RNA synthesis and in vitro translation
The 1941 bp open reading frame of mouse PLC was cloned into pCR-Blunt II-TOPO, sequenced and subcloned (pTarget, Promega) to generate pTarget-mPLC. Complementary RNA (cRNA) was synthesised from linearised pTarget-mPLC (Ribomax RNA synthesis, Promega) in the presence of 3 mM m7G(5')ppp(5')G, isopropanol precipitated and resuspended in DEPC-treated water containing 4 U/µl RNasin (Promega). Mutagenesis of 210Asp to 210Arg in PLC to produce D210RPLC was achieved using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Constructs and cRNAs for rat PLC1 and 
PHPLC1, which encoded the full-length (756 amino acids) and PH domain-deleted PLC1 (1-132), respectively, and D210RPLC were produced in pTarget as above. cRNA (2 µg) was expressed in vitro (Reticulocyte lysate system, Promega) in the presence of [35S]methionine (Amersham Pharmacia). Radiolabelled protein, analysed by SDS-PAGE and autoradiography, was displayed using QuantityOne software (BioRad).
Epitope tagging, bacterial expression and PLC quantitation
The 1941 bp open reading frame of mouse PLC was subcloned into pGBK-T7 (Clontech) with an in-frame Myc epitope tag at the 5'-end. The Myc-PLC was further subcloned into pcDNA3.1 and sequence-verified before cRNA synthesis from the T7 site (Ribomax) for egg microinjection, as described above. For bacterial expression, Myc-PLC was subcloned into pBAD (Invitrogen) with an in-frame hexahistidine tag at the 3' end. The Myc-PLC-Histag protein was produced in 0.2% w/v arabinose-induced, BL21(DE3)pLysS E. coli, after extraction of the pelleted bacteria by five freeze-thaw and ultrasonication cycles, then purified by nickel affinity chromatography (ProBond, Invitrogen). Protein quantitation was performed using the BCA protein assay (Pierce).
Densitometric analysis of the Myc-PLC band expressed in eggs microinjected with different cRNA concentrations (Fig. 6C), Myc-PLC-Histag protein purified from E. coli, and calibrated sperm extract PLC derived from 104-106 mouse sperm, employed a Myc monoclonal antibody (1:2000, Santa Cruz Biotechnology) and rabbit anti-PLC antiserum (1:1000), respectively, using QuantityOne software (BioRad). A calibration standard plot, from analysis by immunoblot densitometry (Malek et al., 1997) using the Myc antibody, was constructed using defined amounts of Myc-PLC-Histag protein, purified from E. coli, to enable the calculation of the relative Myc-PLC content in batches of 100 microinjected eggs. For the quantitation analysis, expression of the Myc-PLC protein was assumed to be linear with time after cRNA microinjection, as was shown for microinjected EGFP cRNA expressed in mouse eggs (Aida et al., 2001). This assumption was necessary because the c-Myc-PLC protein was below the detection limit within 3 hours of cRNA microinjection (data not shown). Hence, for a single mouse egg, the calculated 440-750 fg of Myc-PLC protein expressed 5 hours after microinjection with 0.02 mg/ml cRNA, was equivalent to 44-75 fg expressed at 0.5 hours, the time when the first Ca2+ transient is normally observed (Fig. 5B). A separate calibration plot using the anti-PLC antibody was constructed with different Myc-PLC-Histag protein concentrations to enable estimation of the relative PLC content in defined numbers of mouse sperm (Fig. 6D).
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from sperm extracts) prepared from hamster sperm were incubated for 1 hour at 4°C with control IgG or anti-PLC. antibody that had been covalently attached to Protein G beads (1 mg/ml, Seize X Kit, Pierce). The PLC content of the supernatant and precipitated beads was determined by immunoblot analysis with anti-PLC antibody. Antibody-treated sperm supernatants were also analysed for Ca2+ release activity by fluo-3 fluorometry with sea urchin egg homogenates, as described above, and for ability to generate Ca2+ oscillations by microinjection into mouse eggs, as described below. Maximal immunodepletion of the sperm PLC protein was achieved by using an optimised ratio of antibody beads to sperm extract for each experiment (n=4). The optimal ratio was empirically determined for each sperm extract preparation as the minimum concentration of sperm extract (0.3-0.8 mg/ml) that still retains Ca2+ release activity after treatment with the control IgG beads.
Preparation and handling of gametes
Mouse egg procedures were carried out either in Hepes-buffered KSOM or amino acid supplemented KSOM (Summers et al., 2000). Female MF1 mice were superovulated by injection with 5 IU of PMSG followed 48 hours later by HCG (Intervet). Eggs were collected 13.5-14.5 hours after HCG, maintained in 100 µl droplets of H-KSOM under mineral oil at 37°C and cRNA microinjection performed within 1 hour. Expression of Myc-PLC in eggs was examined 5 hours after cRNA microinjection, by adding SDS sample buffer to pelleted eggs and incubating at 95°C for 5 minutes prior to SDS-PAGE, immunoblot then densitometric analysis with the Myc monoclonal antibody, as described above. Calibrated mouse sperm pellets were resuspended in 10 mM Tris-HCl pH 7.5, 15 mM dithiothreitol (Perry et al., 1999), then subjected to five freeze-thaw cycles in liquid N2 and centrifuged at 20,000 g at 4°C for 10 minutes, before densitometric analysis of the soluble extract with PLC antibody, as described above. For in vitro fertilisation studies, sperm were capacitated for 2-3 hours before being added to eggs. Egg activation and development studies were in H-KSOM containing 2 µM cytochalasin D for 4 hours. Further development to two-cell stage, morula and blastocyst stage was carried out in 50 µl droplets of KSOM under mineral oil at 37°C in a 5% CO2 incubator.
Measurement of intracellular Ca2+ in MII-arrested mouse eggs
Eggs loaded with 4 µM Fura red-AM (Molecular Probes) for 10 minutes were washed in H-KSOM and placed on a Nikon Diaphot stage. Loading media included sulfinpyrazone to prevent dye compartmentalisation and extrusion (Lawrence et al., 1997). cRNA solutions in 120 mM KCl, 20 mM Hepes, pH 7.4, were microinjected to 3-5% of egg volume as previously described (Swann, 1990). Protein synthesis was inhibited in control experiments (Lawrence et al., 1998; Jones et al., 1995) where eggs were preincubated in solution containing 10 µM cycloheximide for 30 minutes before microinjection with PLC cRNA (0.02 mg/ml; n=9). Injection volume was estimated from the displacement caused by bolus injection. Ca2+ measurements were performed on a CCD-based imaging system as previously described (Lawrence et al., 1997), or a Zeiss Axiovert 100 with illumination from a monochromator (Photonics) controlled by MetaFluor v4.0 (Universal Imaging Corp).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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70 kDa) in immunoblots of mouse, boar and hamster sperm (Fig. 1A). Soluble protein extracts from several other tissues are devoid of this immunoreactivity, suggesting that the 70 kDa protein is specifically enriched in sperm (Fig. 1A). Gel filtration chromatography of sperm extracts shows that the immunoreactive sperm protein elutes between the 150 kDa and 29 kDa markers, consistent with a 70 kDa monomer in solution (Fig. 1B). Importantly, the 70 kDa protein specifically co-migrates with Ca2+ release activity in fluorometric assays using egg homogenate (Fig. 1B). This is in contrast to previous chromatographic studies where antibodies to the PLCß, and isoforms showed that they did not co-migrate with Ca2+ releasing activity (Wu et al., 2001; Parrington et al., 2002). The elution profile further indicates that the 70 kDa sperm protein is unrelated to the recently discovered PLC
, which has a molecular mass of 250 kDa (Lopez et al., 2001; Song et al., 2001), and therefore it could be a new PLC.
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2.2 kb sequence, untranslated regions at the 5' and 3' ends, of 194 basepairs (bp) and 52 bp (excluding polyA+-tract), respectively, are found flanking a single open reading frame (ORF) of 1941 bp. The ORF encodes a novel protein sequence of 647 amino acids, with a predicted molecular mass of 74 kDa and pI of 5.3 (Fig. 2A). The novel 74 kDa protein includes the C-terminal peptide sequence used to produce the antiserum and is consistent with the native sperm protein of 70 kDa detected in immunoblots (Fig. 1A). Blastp sequence analysis suggests that the sperm protein is a novel PLC isoform, smaller than all those previously identified (PLCß, , and ) (Katan and Williams, 1997; Rebecchi and Scarlata, 1998; Rhee, 2001), which we accordingly assign PLC.
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with PLC1 reveals that the most notable difference of this new isoform is that it lacks an N-terminal PH domain (Fig. 2A,B). A single PH domain is found at the N terminus of all the PLCß, and isoforms, and the PH domain of PLC1 has been shown to be involved in membrane phospholipid interactions (Katan and Williams, 1997; Rebecchi and Scarlata, 1998; Rhee, 2001). The sequence analysis also indicates that PLC possesses the typical X and Y catalytic domains found in all known PLCs (residues 168-307 and 386-502 of PLC, respectively). The X and Y domains are between a tandem pair of N-terminal EF hand-like domains and a C-terminal C2 domain (residues 20-150 and 521-625, respectively), both of which are present in most PLCs (Fig. 2B). The X and Y domains of PLC contain the PLC1 active site residues, corresponding to 178His, 210Asp and 223His, that have been shown to be involved in catalysis by site-directed mutation studies of PLC1 (Ellis et al., 1993; Ellis et al., 1998) and are conserved across the entire PLC family (Katan, 1998; Rebecchi and Pentyala, 2000). Another distinction between PLC and PLC1 is the extended X-Y linker sequence in PLC (residues 308-385), which has a high proportion of charged residues (Fig. 2A). The X-Y linker region is the only part of PLC1 that was not determined in the 3D crystal structure (Williams, 1999). Multiple alignment of PLC with the other mammalian PLC isoforms shows that it has the highest degree of similarity with the PLC group (33% identity with PLC1) and the lowest with PLC (9% identity; Fig. 2C). The classification of PLC as a distinct isoform is supported by phylogeny analysis of the twelve identified mammalian PLCs, which suggests that is the least divergent PLC isoform from a hypothetical precursor, with the rank order <<ß<< (Fig. 2D). In accordance with this observation, the domain structure of is similar to plant PLCs that also lack an N-terminal PH domain but retain normal enzymatic properties (Rebecchi and Pentyala, 2000). No plant PLCs with domain structures of the mammalian ß, , or isoforms have been identified (Rebecchi and Pentyala, 2000).
Northern blot analysis with mouse tissue mRNAs shows that PLC is present as a relatively abundant 2.3 kb transcript only in the testis (Fig. 3A). The transcript abundance is consistent with the significant number of mouse testis ESTs found in the database. The transcript size of 2.3 kb for PLC matches the spermatid cDNA clone with a 1941 bp ORF plus 300 bp of untranslated sequence (Fig. 2A). The PLC transcript distribution also is congruent with immunoblot analysis of a panel of mouse tissues which suggests testis-specificity, as PLC protein expression is not detected in any sample other than sperm (Fig. 3B). Sperm cell-specificity of PLC expression within testis was examined by performing PCR on cDNA from mouse spermatids and mouse testis devoid of spermatids. PLC amplification is observed with spermatid cDNA but not with testis cDNA devoid of spermatids (Fig. 3C), suggesting that PLC expression within testis is sperm cell-specific. No PLC isoform has previously been found to be sperm specific, although a splice variant of PLC4 enriched in testis (Nagano et al., 1999) was shown to be involved in the zona pellucida-induced acrosome reaction (Fukami et al., 2001).
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triggers Ca2+ oscillations in eggs; Fissore et al., 1998). To examine whether sperm PLC could trigger such Ca2+ oscillations, we introduced PLC complementary RNA (cRNA) by microinjection into MII-arrested mouse eggs, as described previously for spermatogenic cell mRNA (Parrington et al., 2000). Eggs microinjected with a pipette concentration of 2 mg/ml PLC cRNA, corresponding to <0.1 mg/ml in the egg after a 3-5% injection volume, underwent a prolonged series of Ca2+ oscillations that commence within 15-20 minutes (Fig. 4A, top trace). The high oscillation frequency is similar to that observed upon microinjection of concentrated sperm extracts into mouse eggs (Tang et al., 2000). Ca2+ oscillations of similar amplitude, but lower frequency, are obtained with a 1000-fold dilution to 0.002 mg/ml PLC cRNA (Fig. 4A, middle trace; 0.0001 mg/ml in egg). None of the eggs treated with cycloheximide to block protein synthesis showed any Ca2+ transients after PLC cRNA-microinjection (0.02 mg/ml, n=9; Fig. 4A, bottom trace). Robust Ca2+ oscillations were observed in 100% of the eggs microinjected with the four different PLC cRNA concentrations tested, ranging from 0.002-2 mg/ml (Fig. 4B). Importantly, the frequency, but not the amplitude, of Ca2+ oscillations varies with PLC cRNA concentration, directly matching the same phenomenon observed with different concentrations of sperm extract (Swann, 1990). The highest pipette concentration used, 2 mg/ml, produces Ca2+ oscillations with a mean interspike interval of 7.3±3.2 minutes (Fig. 4B). The lowest pipette concentration of PLC cRNA that gives oscillations within 2 hours of injection (0.002 mg/ml), displayed a mean interspike interval of 20.1±5.4 minutes (Fig. 4B). Both of these values are significantly different to the mean interspike interval produced with in vitro fertilisation (IVF) of mouse eggs (12.1±5.8 minutes). However, the interspike intervals for 0.2 and 0.02 mg/ml PLC cRNA (13.6±3.2 and 12.7±6.0 minutes, respectively) are not significantly different from IVF (Fig. 4B).
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) display some unique features. The first Ca2+ transient invariably lasts longer than subsequent oscillations (Fig. 5A), and exhibits a set of intriguing, smaller sinusoidal increases on top of the main peak (Fig. 5A-I). Microinjection of a pipette concentration of PLC cRNA that produces an interspike interval matching IVF (i.e. 0.02 mg/ml; Fig. 4B) results not only in the same, longer initial Ca2+ transient, but also displays a similar pattern of smaller sinusoidal increases (Fig. 5B and Fig. 5B-I). The first Ca2+ increase after 0.02 mg/ml PLC cRNA microinjection matches the first IVF transient in both average duration (PLC 2.8±0.6 minutes, n=39 versus IVF 3.0±0.7 minutes, n=16), and also in reproducibly producing the cluster of smaller Ca2+ increases superimposed on the first transient (Fig. 5B-I). A concentration of 0.02 mg/ml PLC cRNA was used for subsequent microinjection experiments, unless stated otherwise, to provide the precise Ca2+ signalling conditions that are stereotypical of fertilisation.
The ability of sperm PLC to initiate Ca2+ oscillations in eggs is specific to this novel PLC isoform because microinjecting a PLC cRNA (2 mg/ml), structurally the most similar mammalian isoform to PLC (Fig. 2B), does not trigger a Ca2+ increase in any of the 14 eggs tested (Fig. 5C). The lack of effect of PLC1 cRNA is consistent with the inability of microinjected PLC1 protein to cause any Ca2+ changes in mouse eggs (Jones et al., 2000). As the lack of an N-terminal PH domain is the most distinctive difference between PLC and PLC1 (Fig. 2B), the function of PLC could possibly be mimicked by a truncated PLC1 without the PH domain. Therefore, a deletion construct of PLC1 minus the N-terminal 132 residue PH domain (PHPLC1) was prepared, resembling the domain structure of sperm PLC. Microinjection of PHPLC1 cRNA into eggs does not result in any detectable Ca2+ changes (Fig. 5D, 2 mg/ml, n=12 eggs), suggesting that additional factors unique to sperm PLC are crucial for Ca2+ mobilisation in mammalian eggs. To determine whether catalytically active sperm PLC is required for Ca2+ mobilisation in the egg, cRNA with a mutation at 210Asp, a putative active site residue critical for PLC enzyme function (Katan, 1998; Williams, 1999; Rebecchi and Pentyala, 2000), was microinjected. Mutation of the corresponding residue in PLC1, 343Asp to 343Arg, was shown to be the most severe of numerous site-directed alterations, causing a 180,000-fold reduction in PIP2-mediated hydrolysis of PLC1 (Ellis et al., 1998). The microinjection of D210RPLC cRNA into eggs, even at high concentration (2 mg/ml), does not produce any Ca2+ increase (Fig. 5E, n=22). This suggests that 210Asp is crucial for PLC enzyme activity, and that IP3 production is necessary for Ca2+ release to occur in eggs (Miyazaki et al., 1983; Stricker, 1999; Brind et al., 2000; Jellerette et al., 2000). The four different cRNAs used in this study, PLC, D210RPLC, PHPLC1 and PLC1, were also expressed in vitro in rabbit reticulocyte lysates, illustrating that they are correctly synthesised and yield the predicted protein sizes of 74, 74, 70 and 85 kDa, respectively (Fig. 5F).
Physiological level of PLC in a single sperm
The sperm factor hypothesis predicts that a single sperm contains sufficient activating factor to initiate Ca2+ release upon sperm-egg fusion (Swann, 1990; Stricker, 1999). The observation of sperm PLC cRNA triggering fertilisation-like Ca2+ oscillations in eggs (Figs 4 and 5) is of physiological significance only if the PLC protein expressed in a single egg is similar to the native PLC present in a single sperm. In order to quantitate the PLC expressed in microinjected eggs, a Myc epitope tag was introduced at the N terminus of PLC (Lopez et al., 2001). Microinjected Myc-PLC cRNA at different concentrations is as effective at generating Ca2+ oscillations in eggs (Fig. 6A, 0.02 mg/ml) as the untagged PLC (Fig. 4B), indicating that the N-terminal attachment of the Myc tag is not deleterious to PLC activity, as was shown for Myc-PLC (Lopez et al., 2001). Furthermore, the Myc-PLC protein expressed in eggs is readily detected in immunoblots using an anti-Myc monoclonal antibody, as a single band with the predicted mass of 78 kDa, whereas uninjected eggs exhibit no immunoreactivity (Fig. 6B). Comparison of the relative mobility of native mouse sperm PLC (Fig. 6c, 74 kDa) and recombinant Myc-PLC protein [Fig. 6C, 78 kDa (74 kDa PLC + 4 kDa Myc tag)] indicates that the deduced ORF of the PLC cDNA clone (Fig. 2A, 74 kDa) represents the complete sperm PLC sequence. Densitometric analysis of the immunoreactive 78 kDa Myc-PLC protein expressed in eggs (Fig. 6C, 100 eggs microinjected with each Myc-PLC cRNA concentration), compared with calibrated amounts of purified recombinant Myc-PLC protein produced in bacteria, enabled the determination of 44-75 fg/egg (n=4) as the amount of PLC protein that triggers Ca2+ oscillations using 0.02 mg/ml cRNA (see Materials and Methods). This cRNA concentration is the one that most closely mimics the IVF response, though tenfold lower levels (i.e. 4-8 fg PLC protein/egg using 0.002 mg/ml cRNA) are also able to cause Ca2+ oscillations (Fig. 4).
The PLC content of sperm was also determined by densitometry with a PLC polyclonal antibody using a defined number of mouse sperm and compared with calibrated amounts of recombinant PLC protein (Fig. 6D). Using densitometric values within the recombinant PLC protein calibration plot, obtained from samples comprising 104-106 mouse sperm, a single mouse sperm was calculated to contain 20-50 fg PLC protein (n=4). The level of PLC able to produce Ca2+ oscillations in a single egg similar to fertilisation (4-75 fg, i.e. with 0.002-0.02 mg/ml cRNA) is therefore in the same range as the single sperm content of PLC (20-50 fg). The observed quantitative correlation indicates that the PLC from a single sperm is sufficient to produce the Ca2+ oscillations observed upon sperm-egg fusion.
Sperm PLC depletion abrogates Ca2+ oscillations
The features of sperm PLC at the functional (Figs 4 and 5) and quantitative (Fig. 6) level are fully consistent with characteristics observed for the sperm factor present in mammalian sperm extracts (Swann, 1990; Berrie et al., 1996; Wu et al., 1997; Stricker, 1997; Wu et al., 1998; Jones et al., 1998b; Kyozuka et al., 1998; Tang et al., 2000). However, it remains possible that sperm components other than PLC are also involved in causing Ca2+ release in eggs. To address whether the PLC in sperm is uniquely responsible for Ca2+ mobilisation in eggs, the PLC content of sperm extracts was specifically depleted using an anti-PLC antibody. Immunoblot analysis indicates that sperm extract supernatant retains the PLC protein after control antibody treatment, in contrast to PLC antibody-treated supernatant where the PLC is absent (Fig. 7A, S– and S+, respectively). Analysis of the corresponding precipitated antibody samples reveals that the sperm PLC is effectively removed by PLC antibody, but not by the control antibody (Fig. 7A, P+ and P–, respectively). Assessment of Ca2+ release activity in antibody-treated sperm extracts using sea urchin egg homogenate assays shows that PLC-depleted samples lack any Ca2+ mobilising activity, whereas a robust Ca2+ release is observed with the control antibody-treated sperm extract containing PLC protein (Fig. 7B, S+ and S–, respectively). Moreover, microinjection of antibody-treated sperm extracts into mouse eggs illustrates that the ability of untreated samples to generate IVF-like Ca2+ oscillations (Fig. 7C, top trace) is fully preserved in control antibody-treated samples (Fig. 7C, second trace, n=13), while PLC-depletion effectively abrogates Ca2+ release activity (Fig. 7C, bottom two traces, n=13). These PLC antibody depletion experiments (n=4) suggest that PLC is the sole component of sperm extracts possessing the ability to cause Ca2+ release in mouse eggs. Taken together with evidence that the PLC level in a single mouse sperm is sufficient to trigger IVF-like Ca2+ oscillations in a single mouse egg (Figs 4 
-6), the immunodepletion data provides compelling evidence that PLC is synonymous with the previously described mammalian sperm factor (Swann, 1990; Berrie et al., 1996; Wu et al., 1997; Stricker, 1997; Wu et al., 1998; Jones et al., 1998b; Kyozuka et al., 1998; Tang et al., 2000).
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activates normal embryo development; Kline and Kline, 1992). Microinjection of sperm extract into eggs also produces activation and the consequent cellular processes leading to embryo development (Stice and Robl, 1990; Fissore et al., 1998; Sakurai et al., 1999). The isolated sperm factor molecule therefore is predicted to support embryo development after egg activation, providing a crucial test for any putative sperm factor candidate (Fissore et al., 1998). Because eggs that are microinjected with PLC cRNA (0.02mg/ml) display all the properties of Ca2+ oscillations indistinguishable from those of IVF (Fig. 4B, Fig. 5B) and is equivalent to the PLC content of a single sperm (Fig. 6), their ongoing development was monitored for several days after PLC-microinjection. PLC-microinjected eggs underwent activation (Fig. 8A) because normal development proceeds to the two-cell stage within 24 hours (78%, n=147), and many reach the morula or blastocyst stages by 4-5 days (62%, n=76). None of the eggs microinjected with buffer control reach the two-cell stage, indicating activation as an artefact of microinjection procedure has not occurred (data not shown). The proportion of PLC-induced embryos that develop to either the two-cell, or morula and blastocyst stages, is the same as for eggs that are either parthenogenetically activated (Bos-Mikich et al., 1997) by strontium ions (n=75), or when embryos are collected at the one-cell stage from female mice after in vivo fertilisation (n=101) upon mating with males (Fig. 8A). Photomicrographs taken at 24 hours and 5 days after PLC-microinjection into mouse eggs show the appearance of normal embryo development to the two-cell stage and blastocyst stage (left and right panel, respectively, Fig. 8B). There are no morphological differences to embryos obtained after fertilisation with sperm (data not shown). Thus, after inducing Ca2+ oscillations in the egg, sperm PLC-microinjection also triggers the entire cascade of events required for activation and embryo development, in the same manner as sperm at fertilisation.
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other than PIP2 hydrolysis is responsible for egg activation, such as a protein-protein interaction with a distinct egg molecule. To test whether an enzymatically active PLC is required for egg activation and embryo development, the D210RPLC cRNA (0.02mg/ml), which has been shown to be defective in triggering Ca2+ oscillations (Fig. 5E) (Ellis et al., 1998; Katan, 1998; Williams, 1999; Rebecchi and Pentyala, 2000), was microinjected and egg activation assessed after 24 hours. None of the D210RPLC cRNA-microinjected eggs were found to proceed to the pronuclear or two-cell stage (Fig. 8C, n=20), suggesting that the enzymatic function of sperm PLC is crucial for egg activation. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, as the original observation of Ca2+ oscillations at mammalian fertilisation (Cuthbertson and Cobbold, 1985), the molecular mechanism has remained an enigma. A popular model of Ca2+ signalling at fertilisation involves a sperm surface ligand interacting with a receptor on the egg plasma membrane. The ligand-bound membrane receptor couples with an egg PLC to stimulate IP3 production and Ca2+ release, analogous to the signalling pathway found ubiquitously in somatic cells (Berridge et al., 2000). However, the egg and sperm molecules required for the operation of this ‘receptor’ model have not been identified despite extensive studies (Stricker, 1999).
The second major hypothesis involves a sperm cytosolic protein that enters the egg and causes Ca2+ release (Swann, 1996; Yamamoto et al., 2001). This ‘sperm factor’ model, though hindered by initial quandaries (Parrington et al., 1996; Sette et al., 1997), has gained increasing credence due to the numerous studies demonstrating the potency of sperm extracts in effecting Ca2+ release in eggs (Stice and Robl, 1990; Swann, 1990; Nakano et al., 1997; Stricker, 1997; Wu et al., 1997; Jones et al., 1998b; Kyozuka et al., 1998; Wu et al., 1998; Parrington et al., 1999; Dong et al., 2000; Jones et al., 2000; Rice et al., 2000; Tang et al., 2000; Wu et al., 2001; Yamamoto et al., 2001; Parrington et al., 2002). Moreover, the sperm factor model is congruous with the amount of activity contained in a single sperm (Nixon et al., 2000) and is further supported by the technique of intracytoplasmic sperm injection (ICSI), a clinically effective IVF procedure that has produced thousands of live births (Bonduelle et al., 1999). In the ICSI method, which bypasses the possibility of sperm-egg membrane interaction, a single spermatozoa is injected directly into a human egg to cause Ca2+ oscillations, activation and development to term (Bonduelle et al., 1999; Yanagida et al., 2001). Interestingly, the ICSI practice of breaking off the sperm tail to enhance the rate of egg activation (Dozortsev et al., 1995; Yanagida et al., 2001) could be explained by the facilitated release of sperm cytosolic contents, including the sperm factor.
These two major models for Ca2+ signalling at fertilisation have developed an overlap following recent observations indicating that a PLC activity is indeed involved in triggering Ca2+ oscillations, but the PLC is in the sperm, not the egg (Jones et al., 1998b; Rice et al., 2000; Wu et al., 2001; Parrington et al., 2002). However, comprehensive analysis of known PLC isoforms by different approaches have all concluded that none of them could be the sperm factor (Mehlmann et al., 1998; Jones et al., 2000; Fukami et al., 2001; Mehlmann et al., 2001; Wu et al., 2001; Parrington et al., 2002). There remains the possibility of an undiscovered sperm PLC with the requisite Ca2+ signalling properties, and this was directly addressed in the present study.
The revelation of abundant testis-derived ESTs with PLC homology led to our characterisation of a novel sperm PLC isoform (Fig. 1). The new isoform, PLC, is the smallest PLC identified to date, most closely resembling the PLC class, but without an N-terminal PH domain and a longer X-Y domain linker sequence (Fig. 2). The tissue transcript and protein expression profile indicates sperm-specific enrichment of the PLC protein, consistent with a gamete-specific role (Fig. 3). Functional analysis by expression in mammalian eggs provides exquisite evidence that PLC possesses the mandatory properties of the sperm factor. PLC exhibits the unique ability to produce Ca2+ oscillations with the characteristic interspike interval (Fig. 4), and the intriguing, first transient profile-specificity (Fig. 5), found in Ca2+ signalling at fertilisation. The inability of the PH-domain-deleted PLC1 to mimic fertilisation Ca2+ transients, suggests an exclusive functional specificity for the sperm PLC domains inside mammalian eggs (Fig. 5). Similarly, the functionally ineffective PLC with a catalytic site mutation (Fig. 5) is consistent with the previously shown vital role of a PLC and IP3 production in mobilising Ca2+ in eggs (Miyazaki et al., 1993; Brind et al., 2000; Jellerette et al., 2000). Quantitative correlation of the PLC level that produces an IVF-like Ca2+ response with that found in a single sperm (Fig. 6), together with demonstration of the unique role of the PLC within sperm extracts in effecting Ca2+ release in eggs (Fig. 7), directly support the tenet that sperm PLC has a physiologically relevant role in egg activation. Furthermore, the normal development of PLC-microinjected eggs to the blastocyst stage (Fig. 8) shows that Ca2+ oscillations, which are triggered solely via PLC, are both necessary and sufficient to initiate the entire network of cellular processes that operate from egg activation through early embryo development to blastocyst. These decisive features of PLC argue that it is an important component of the augured mammalian sperm factor and also that there is a physiological role for PLC in egg activation and embryo development during mammalian fertilisation.
Discovery of PLC as a novel mediator of intracellular Ca2+ regulation will enable an increased understanding of the propagation mechanism of large amplitude, low-frequency cytosolic Ca2+ oscillations (Berridge et al., 2000). Identification of PLC as a component of the putative physiological sperm factor should help to reveal the molecular mechanisms involved in subsequent stages of embryo development after egg activation. Analysis of human sperm PLC may also provide a new framework for understanding some cases of male factor infertility where the sperm are ineffective in stimulating development (Rybouchkin et al., 1996; Battaglia et al., 1997). Finally, PLC could be applied in approaches to improve egg activation rates, for example, after somatic cell nuclear transfer into enucleated eggs, in the production of stem cells for therapy of human diseases (Aldhous, 2001).
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ACKNOWLEDGMENTS |
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1 plasmid by M. Katan. We are grateful for the advice and encouragement of M. J. Berridge, L. K. Borysiewicz and D. R. Trentham. |
REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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