PubMed Health⌕ Search

Biomedical subjects

J Parrington

Publications and source records attributed to J Parrington.

At least 19 recordsLinked to original sources

Phospholipase Czeta, the trigger of egg activation in mammals, is present in a non-mammalian species.

The activation of the egg to begin development into an embryo is triggered by a sperm-induced increase in intracellular egg Ca2+. There has been much controversy about how the sperm induces this fundamental developmental event, but recent studies suggest that, in mammals, egg activation is triggered by a testis-specific phospholipase C: PLCzeta. Since the discovery of PLCzeta, it has been unclear whether its role in triggering egg activation is common to all vertebrates, or is confined to mammals. Here, we demonstrate for the first time that PLCzeta is present in a non-mammalian vertebrate. Using genomic and cDNA databases, we have identified the cDNA encoding a PLCzeta orthologue in the domestic chicken that, like the mammalian isoforms, is a testis-specific gene. The chicken PLCzeta cDNA is 2152 bp in size and encodes an open reading frame of 639 amino acids. When injected into mouse oocytes, chicken PLCzeta cRNA triggers Ca2+ oscillations, indicating that it has functional properties similar to those of mammalian PLCzeta. Our findings suggest that PLCzeta may have a universal role in triggering egg activation in vertebrates.

Amino Acid Sequence↗

The NAADP receptor: commentary on Billington et al.

NAADP is a recently described calcium-mobilizing messenger. First discovered as a potent calcium-releasing molecule in sea urchin eggs, its actions have now been reported in several mammalian cell types. In the sea urchin egg, NAADP-sensitive calcium release channels appear distinct from inositol trisphosphate or ryanodine receptors, and are mainly localized to acidic compartments. In this study, Billington et al. extend the pharmacology of the putative NAADP receptor utilizing molecules unrelated to NAADP itself. This work may provide an important step in developing selective NAADP receptor modulators that will help define the role of NAADP in cell signalling.

Animals↗

Phospholipase C isoforms in mammalian spermatozoa: potential components of the sperm factor that causes Ca2+ release in eggs.

Injection of a soluble protein factor from mammalian spermatozoa triggers Ca2+ oscillations in mammalian eggs similar to those seen at fertilization. This sperm factor also generates inositol 1,4,5-trisphosphate and causes Ca2+ release in sea urchin egg homogenates and frog eggs. Recent studies have indicated that the sperm factor may be an inositol-specific phospholipase C (PLC) activity. This study investigated whether any of the commonly known PLC isoforms are components of the sperm factor. PLCbeta, PLCgamma and PLCdelta isoforms were shown to be present in boar sperm extracts. However, upon column fractionation of sperm extracts, none of the PLC isoforms detected correlated with the ability to cause Ca2+ release in eggs. In addition to our previous work on recombinant PLCs, it was also shown that PLCdelta3, PLCdelta4 and its splice variant PLCdelta4 Alt1 fail to cause Ca2+ release. The recently discovered 255 kDa PLCepsilon isoform also appears unlikely to be a component of the sperm factor, as fractionation of sperm extracts on a gel filtration column demonstrated that the peak of Ca2+-releasing activity was associated with fractions of 30-70 kDa. These findings indicate that the sperm factor that triggers Ca2+ release in eggs does not appear to have a known PLC isoform as one of its components.

Animals↗

Potential role of a sperm-derived phospholipase C in triggering the egg-activating Ca2+ signal at fertilization.

An increase in intracellular Ca2+ at fertilization is the trigger for egg activation in all species that have been studied. Exactly how sperm-egg interaction leads to this Ca2+ increase has not been established. There is increasing support for the hypothesis that the spermatozoon introduces a Ca2+-releasing protein into the egg cytoplasm after gamete membrane fusion. This review discusses the merits of this 'sperm factor' hypothesis and presents evidence indicating that the sperm factor, at least in mammals, consists of a phospholipase C with distinctive properties. This evidence leads us to propose that, after gamete fusion, a sperm-derived phospholipase C causes production of inositol 1,4,5- trisphosphate, which then generates Ca2+ waves from within the egg cytoplasm.

Animals↗

Mammalian sperm contain a Ca(2+)-sensitive phospholipase C activity that can generate InsP(3) from PIP(2) associated with intracellular organelles.

We have previously described a phospholipase C (PLC) activity in mammalian sperm cytosolic extracts. Here we have examined the Ca(2+) dependency of the enzyme, whether there is enough in a single sperm to account for Ca(2+) release at fertilization, and finally where in the egg is the phosphatidyl 4,5-bisphosphate, the substrate for the enzyme. As for all PLCs examined so far in vitro, we found that the boar sperm PLC activity was Ca(2+) dependent. Specific activity increased when free Ca(2+) levels were micromolar. However, even at nanomolar free Ca(2+) concentration the boar sperm PLC activity was considerable, being two orders of magnitude greater than PLC activities in other tissues. We calculated that PLC activity of a single boar sperm in a mammalian egg is enough to generate 400 nM inositol 1,4,5-trisphosphate (InsP(3)) in 1 min, which may be sufficient to account for the observed Ca(2+) changes in an egg at fertilization. We fractionated sea urchin egg homogenate and examined the ability of boar sperm extract to generate InsP(3) from these fractions. The sperm PLC activity triggered InsP(3) production from a PIP(2)-enriched nonmicrosomal egg compartment that contained yolk platelets. We propose that this sperm PLC activity, which is active at nanomolar Ca(2+) levels and hydrolyzes PIP(2) from intracellular membranes, could be involved in the Ca(2+) changes observed at fertilization.

Aniline Compounds↗

Different Ca2+-releasing abilities of sperm extracts compared with tissue extracts and phospholipase C isoforms in sea urchin egg homogenate and mouse eggs.

A soluble phospholipase C (PLC) from boar sperm generates InsP(3) and hence causes Ca(2+) release when added to sea urchin egg homogenate. This PLC activity is associated with the ability of sperm extracts to cause Ca(2+) oscillations in mammalian eggs following fractionation. A sperm PLC may, therefore, be responsible for causing the observed Ca(2+) oscillations at fertilization. In the present study we have further characterized this boar sperm PLC activity using sea urchin egg homogenate. Consistent with a sperm PLC acting on egg PtdIns(4,5)P(2), the ability of sperm extracts to release Ca(2+) was blocked by preincubation with the PLC inhibitor U73122 or by the addition of neomycin to the homogenate. The Ca(2+)-releasing activity was also detectable in sperm from other species and in whole testis extracts. However, activity was not observed in extracts from other tissues. Moreover recombinant PLCbeta1, -gamma1, -gamma2, -delta1, all of which had higher specific activities than boar sperm extracts, were not able to release Ca(2+) in the sea urchin egg homogenate. In addition these PLCs were not able to cause Ca(2+) oscillations following microinjection into mouse eggs. These results imply that the sperm PLC possesses distinct properties that allow it to hydrolyse PtdIns(4,5)P(2) in eggs.

Animals↗

The application of comparative genomic hybridization to previously karyotyped cervical cancer cell lines.

This investigation is concerned with the application of comparative genomic hybridization (CGH) to DNA from previously fully karyotyped cervical cancer cell lines using G-banding and fluorescence in situ hybridization (FISH) to compare the chromosome copy numbers observed in karyotypes with the profile shifts seen in CGH analysis. It has demonstrated that diploid DNA can be used as a reference to cohybridize with a test sample of any modal number because of the proportional representation of every chromosome arm and region in equal volumes of both test and reference DNAs. Profile shifts in the near-diploid line gave a clear indication of over and under-representation of either the whole or parts of chromosome arms. In near-tetraploid samples, profile shifts, either gain or loss due to copy number changes from four to five, five to six, or four to three were smaller and were not always seen; however, the points of profile shift would have allowed us to work out most of the breakpoints if karyotype information had not been available. The profiles, however, did not provide accurate information on the ploidy status; this would need to be measured by other means for the CGH data to be interpreted correctly. The 3q and 8q gain in all the squamous cell carcinoma cell lines appeared very clearly. Comparative genomic hybridization revealed a new breakpoint at 7q31 which was not detected originally on the karyotype in DE3. A breakpoint on 9q was reassigned on the basis of the profile shift from 9q13 to 9q22 in JE6. Clarification of the origin of a small fragment from chromosome 20 constantly present in JE6 showed it to be 20q22-qter.

Carcinoma, Squamous Cell↗

The soluble mammalian sperm factor protein that triggers Ca2+ oscillations in eggs: evidence for expression of mRNA(s) coding for sperm factor protein(s) in spermatogenic cells.

At fertilisation in mammals the sperm initiates a series of Ca2+ oscillations that activate development. One theory of signalling at fertilisation suggests that the sperm contains a soluble protein factor that causes these Ca2+ oscillations by entering the egg after sperm-egg membrane fusion. This theory is supported by the finding that, in some species, injection of sperm protein extracts into eggs triggers a pattern of Ca2+ oscillations similar to those seen at fertilisation. So far, all the direct evidence for a sperm factor has been based upon the injection of soluble proteins from mature sperm. Here, we demonstrate that injection of mRNA extracted from hamster spermatogenic cells also leads to generation of prolonged Ca2+ oscillations in mouse eggs. The ability of spermatogenic cell mRNA to induce Ca2+ oscillations is dependent upon translation into protein and also appears to be specific to spermatogenic cells since injection of mRNA isolated from somatic tissues into eggs was ineffective. These data support the hypothesis that sperm contain a soluble, cytosolic protein factor that induces Ca2+ oscillations in eggs at fertilisation. These data are discussed in the light of our recent findings that suggest that the sperm factor possesses a phospholipase C activity.

Animals↗

Mechanism of Ca2+ release at fertilization in mammals.

At fertilization in mammals the sperm triggers a series of oscillations in intracellular Ca2+ within the egg. These Ca2+ oscillations activate the development of the egg into an embryo. It is not known how the sperm triggers these Ca2+ oscillations. There are currently three different theories for Ca2+ signaling in eggs at fertilization. One idea is that the sperm acts as a conduit for Ca2+ entry into the egg after membrane fusion. Another idea is that the sperm acts upon plasma membrane receptors to stimulate a phospholipase C (PLC) within the egg which generates inositol 1,4, 5-trisphosphate (InsP(3)). We present a third idea that the sperm causes Ca2+ release by introducing a soluble protein factor into the egg after gamete membrane fusion. In mammals this sperm factor is also referred to as an oscillogen because, after microinjection, the factor causes sustained Ca2+ oscillations in eggs. Our recent data in sea urchin egg homogenates and intact eggs suggests that this sperm factor has phospholipase C activity that leads to the generation of InsP(3). We then present a new version of the soluble sperm factor theory of signaling at fertilization. J. Exp. Zool. (Mol. Dev. Evol.) 285:267-275, 1999.

Animals↗

The soluble sperm factor that causes Ca2+ release from sea-urchin (Lytechinus pictus) egg homogenates also triggers Ca2+ oscillations after injection into mouse eggs.

Cytosolic extracts of boar sperm contain a soluble phospholipase C (PLC) activity that induces Ca2+ release in sea-urchin (Lytechinus pictus) egg homogenates and an uncharacterized protein factor that causes Ca2+ oscillations when injected into mammalian eggs. In the present study we fractionated boar sperm extracts on three different FPLC chromatographic columns and found that the fractions that caused maximal Ca2+ release in sea-urchin egg homogenates were also the ones that triggered Ca2+ oscillations in mouse eggs. Our data suggests that the sperm factor which triggers Ca2+ oscillations in eggs contains a PLC and not the 33 kDa glucosamine deaminase previously suggested to be one its components.

Animals↗

Expression of inositol 1,4,5-trisphosphate receptors in mouse oocytes and early embryos: the type I isoform is upregulated in oocytes and downregulated after fertilization.

A fertilization-induced increase in intracellular Ca2+ is responsible for initiating all of the events of egg activation. In mammals, the Ca2+ increase takes the form of a series of Ca2+ oscillations showing complex temporal and spatial properties. To understand the nature of these changes, we have investigated the expression patterns of the three isoforms of the inositol trisphosphate receptor (InsP3R) during oocyte maturation and preimplantation development. We find that mouse oocytes express mRNAs for all three InsP3R subtypes. Semiquantitative ratio reverse-transcriptase polymerase chain reaction shows that the type II isoform is the predominant message in mature oocytes, representing 67% of the InsP3R mRNA. In contrast, protein analysis reveals that the type I isoform accounts for all of the detectable InsP3R protein, despite representing only 20% of the InsP3R mRNA. The levels of InsP3R protein were examined to determine whether they correlated with the Ca2+ signaling events surrounding the fertilization process. Type I InsP3R protein increased during oocyte maturation and, in addition, within 8 h of fertilization underwent a dramatic decrease. During development to the blastocyst the level of type I InsP3R protein did not return to prefertilization levels and types II and III remained below our detection limit. The decrease in InsP3R protein after fertilization was found to correlate with a decrease in the sensitivity of InsP3-induced Ca2+ release. These studies show that the expression of InsP3R mRNA is developmentally regulated, that Ca2+ signaling at fertilization is mediated exclusively through the type I InsP3R, and that the InsP3R is downregulated after fertilization.

Animals↗

A mammalian sperm cytosolic phospholipase C activity generates inositol trisphosphate and causes Ca2+ release in sea urchin egg homogenates.

Injection of sperm extracts triggers Ca2+ oscillations in mammalian eggs similar to those seen at fertilisation. Here, we show that addition of sperm extracts to sea urchin egg homogenates causes Ca2+ release and inositol 1,4,5-trisphosphate (InsP3) production. Furthermore depleting homogenates of phosphatidylinositol lipids using a phosphatidylinositol-specific phospholipase C blocked the sperm extract from causing InsP3 production and a Ca2+ rise. A response could be recovered by the addition of phosphatidylinositol 4,5-bisphosphate to either sperm extracts or egg homogenates. These data indicate that sperm extracts contain an InsP3-generating phospholipase C which may play a role in Ca2+ release at fertilisation.

Animals↗

The human glucosamine-6-phosphate deaminase gene: cDNA cloning and expression, genomic organization and chromosomal localization.

When mammalian eggs are fertilized by sperm, a distinct series of calcium oscillations are generated which serve as the essential trigger for egg activation and early embryo development. The identification of a soluble hamster sperm 33-kDa protein that co-migrated with calcium oscillation-inducing activity was recently described by Parrington et al. (Parrington, J., Swann, K., Shevchenko, V.I., Sesay, A.K. and Lai, F.A., 1996. Calcium oscillations in mammalian eggs triggered by a soluble sperm protein. Nature 379, 364-368). The hamster sperm 33 kDa protein was termed oscillin because it correlated with calcium oscillation-inducing activity in mammalian eggs. Sequence analysis of the hamster sperm 33 kDa protein indicated no similarity to any known cell signalling molecule, however, it displayed extensive homology with a bacterial glucosamine-6-phosphate deaminase. We have isolated the corresponding human testis homologue of the hamster sperm 33 kDa cDNA. Nucleotide sequence analysis reveals a high level of sequence identity between the hamster and human genes. The deduced protein sequence of the human gene also shares extensive amino acid identity with the bacterial glucosamine-6-phosphate deaminase enzyme. Heterologous expression of the human testis 33 kDa protein produced a glucosamine-6-phosphate deaminase activity. The genomic structure of the human glucosamine-6-phosphate deaminase has been mapped and the gene was localized by fluorescence in situ hybridization (FISH) to chromosome 5q31.

Aldose-Ketose Isomerases↗

Presence and localization of oscillin in human spermatozoa in relation to the integrity of the sperm membrane.

We investigated the presence and localization of oscillin in human spermatozoa in relation to the integrity of the sperm membrane, which was assessed by the hypo-osmotic swelling (HOS) test. We found no gross differences in the presence of oscillin in semen samples from men who presented with 70%, 40%, 25% or 2% of membrane-intact spermatozoa. By immunofluorescence, membrane-intact (HOS-positive) spermatozoa showed staining of a single band at the equatorial region, whereas over 80% of HOS-negative spermatozoa consistently showed a diffuse distribution of oscillin over the sperm head. However, some individuals presented with up to 50% of HOS-positive spermatozoa showing an aberrant localization of oscillin. We found a significant correlation rate (r=0.70, P < 0.05) between the percentage of HOS-positive spermatozoa with an equatorial oscillin localization and the fertilization rates achieved after intracytoplasmic sperm injection. These data suggest that the localization of oscillin in human spermatozoa might have an impact on egg activation and fertilization rates.

Calcium-Binding Proteins↗

Calcium oscillations, sperm factors and egg activation at fertilisation.

When an egg is fertilised by sperm, the first intracellular signalling event observed is a large transient increase in cytoplasmic free Ca2+ ions. Elevated Ca2+ is known to play a vital role as an intracellular messenger in all cells and the Ca2+ signal occurring in the egg at fertilisation triggers the subsequent events that mediate early embryo development. In mammalian eggs, the Ca2+ response is first observed as a Ca2+ wave that initiates near the point of sperm-egg fusion, spreads across the entire egg, and then continues as a series of intracellular Ca2+ oscillations. The way in which the fertilising sperm generates the Ca2+ response in the egg has been the subject of much debate over recent years. One proposal for which there is growing evidence suggests the mechanism of egg activation at fertilisation involves the introduction of a soluble sperm protein into the egg shortly after sperm-egg fusion.

Animals↗

A novel protein for Ca2+ signaling at fertilization.

At fertilization in all species studied the sperm activates the egg by causing an increase in the level of cytoplasmic free Ca2+ concentration. It is still not established how the sperm causes the changes in Ca2+ in the egg, which in the majority of eggs is due to release from internal stores. Current hypotheses about the signaling molecules involved in fertilization are confounded by the fact that for many eggs the fertilization-associated Ca2+ increase is readily mimicked by parthenogenetic activating agents. One exception to this is found for mammalian eggs where there are a series of Ca2+ oscillations observed at fertilization that have distinct characteristics. In this context we discuss three different theories of how sperm trigger Ca2+ release in eggs. We present the case that the sperm mediates its Ca2+ mobilization effects after gamete membrane fusion by introducing a specific protein into the egg cytoplasm. Our argument is based upon the fact that only the mammalian sperm protein factor can trigger a pattern of Ca2+ oscillations that is similar to that induced by the sperm in mammalian eggs. The sperm factor activity is correlated with a novel signaling protein that we have called oscillin and which may mediate Ca2+ release via a novel mechanism.

Animals↗

Molecular and cytological investigations of phosphoglucomutase (PGM1) in the K562 cell line.

Phosphoglucomutase 1 (PGM1) deficiency is a stable characteristic of the erythroleukaemic cell line, K562, whereas the activity of the isozymes of the other two PGM loci (PGM2 and PGM3) is slightly elevated. In this study the molecular basis of PGM1 deficiency was investigated by a combined approach utilising protein electrophoresis, immunodetection, cytogenetic techniques, and DNA and RNA analysis. Isoelectric focusing and activity staining confirmed that K562 has no detectable PGM1 activity. Immunoblot analysis of extracts, separated by isoelectric focusing, starch gel and SDS gel electrophoresis, using monospecific anti-PGM1 antibodies showed that K562 contained no detectable immunoreactive material. Karyotype analysis revealed the presence of two intact chromosomes 1 and a derivative chromosome 1, der(1)t(1;11), each of which carried a copy of the PGM1 gene as demonstrated by fluorescence in situ hybridization using a PGM1 cosmid as probe. Southern blot analysis using a PGM1 cDNA clone as probe suggested that the PGM1 genes had not been subject to any gross structural rearrangements. We were also able to determine that K562 is type PGM1 2+1+ by restriction endonuclease analysis of genomic DNA. Very low levels of PGM1 mRNA which appeared to be full length transcripts were detected in K562 using a reverse transcriptase PCR technique. We conclude that the most likely cause of PGM1 enzyme deficiency in K562 is abnormal regulation of transcription.

Blotting, Southern↗