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

2000 ◽  
Vol 346 (3) ◽  
pp. 743-749 ◽  
Author(s):  
Keith T. JONES ◽  
Miho MATSUDA ◽  
John PARRINGTON ◽  
Matilda KATAN ◽  
Karl SWANN
Keyword(s):  
Phospholipase C ◽  
Sea Urchin ◽  
Tissue Extracts ◽  
Sea Urchin Egg ◽  
Boar Sperm ◽  
Specific Activities ◽  
Mammalian Eggs ◽  
Mouse Eggs

A soluble phospholipase C (PLC) from boar sperm generates InsP3 and hence causes Ca2+ release when added to sea urchin egg homogenate. This PLC activity is associated with the ability of sperm extracts to cause Ca2+ oscillations in mammalian eggs following fractionation. A sperm PLC may, therefore, be responsible for causing the observed Ca2+ 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)P2, the ability of sperm extracts to release Ca2+ was blocked by preincubation with the PLC inhibitor U73122 or by the addition of neomycin to the homogenate. The Ca2+-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 PLCβ1, -γ1, -γ2, -∆1, all of which had higher specific activities than boar sperm extracts, were not able to release Ca2+ in the sea urchin egg homogenate. In addition these PLCs were not able to cause Ca2+ oscillations following microinjection into mouse eggs. These results imply that the sperm PLC possesses distinct properties that allow it to hydrolyse PtdIns(4,5)P2 in eggs.

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

2000 ◽  
Vol 346 (3) ◽  
pp. 743 ◽  
Author(s):  
Keith T. JONES ◽  
Miho MATSUDA ◽  
John PARRINGTON ◽  
Matilda KATAN ◽  
Karl SWANN
Keyword(s):  
Phospholipase C ◽  
Sea Urchin ◽  
Tissue Extracts ◽  
Sea Urchin Egg ◽  
Mouse Eggs

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

1999 ◽  
Vol 341 (1) ◽  
pp. 1-4 ◽  
Author(s):  
John PARRINGTON ◽  
Keith T. JONES ◽  
F. Anthony LAI ◽  
Karl SWANN
Keyword(s):  
Sea Urchin ◽  
Uncharacterized Protein ◽  
Lytechinus Pictus ◽  
Protein Factor ◽  
Chromatographic Columns ◽  
Sea Urchin Egg ◽  
Boar Sperm ◽  
Sperm Factor ◽  
Mammalian Eggs ◽  
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.

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

Reproduction ◽  
2002 ◽  
pp. 31-39 ◽  
Author(s):  
J Parrington ◽  
ML Jones ◽  
R Tunwell ◽  
C Devader ◽  
M Katan ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Sea Urchin ◽  
Soluble Protein ◽  
Gel Filtration ◽  
Protein Factor ◽  
Sea Urchin Egg ◽  
Inositol 1,4,5 Trisphosphate ◽  
Sperm Factor ◽  
Mammalian Eggs ◽  
Mammalian Spermatozoa

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.

scholarly journals Fertilization-Induced Activation of Phospholipase C in the Sea Urchin Egg

1999 ◽  
Vol 215 (2) ◽  
pp. 147-154 ◽  
Author(s):  
Brenda J. Rongish ◽  
Wenjun Wu ◽  
William H. Kinsey
Keyword(s):  
Phospholipase C ◽  
Sea Urchin ◽  
Sea Urchin Egg

PLCζ: a sperm-specific trigger of Ca2+ oscillations in eggs and embryo development

Development ◽  
2002 ◽  
Vol 129 (15) ◽  
pp. 3533-3544 ◽  
Author(s):  
Christopher M. Saunders ◽  
Mark G. Larman ◽  
John Parrington ◽  
Llewellyn J. Cox ◽  
Jillian Royse ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Embryo Development ◽  
Specific Protein ◽  
Normal Embryo ◽  
Egg Activation ◽  
Sperm Factor ◽  
Mammalian Eggs ◽  
Mouse Eggs ◽  
Cytosolic Factor ◽  
Specific Trigger

Upon fertilisation by sperm, mammalian eggs are activated by a series of intracellular Ca2+ oscillations that are essential for embryo development. The mechanism by which sperm induces this complex signalling phenomenon is unknown. One proposal is that the sperm introduces an exclusive cytosolic factor into the egg that elicits serial Ca2+ release. The ‘sperm factor’ hypothesis has not been ratified because a sperm-specific protein that generates repetitive Ca2+ transients and egg activation has not been found. We identify a novel, sperm-specific phospholipase C, PLCζ, that triggers Ca2+ oscillations in mouse eggs indistinguishable from those at fertilisation. PLCζ removal from sperm extracts abolishes Ca2+ release in eggs. Moreover, the PLCζ 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 PLCζ as the molecular trigger for development of a fertilised egg into an embryo.

scholarly journals ADP-ribosyl cyclase: an enzyme that cyclizes NAD+ into a calcium-mobilizing metabolite.

1991 ◽  
Vol 2 (3) ◽  
pp. 203-209 ◽  
Author(s):  
H C Lee ◽  
R Aarhus
Keyword(s):  
Sea Urchin ◽  
Exchange Chromatography ◽  
Reaction Products ◽  
Sea Urchin Eggs ◽  
Tissue Extracts ◽  
Adp Ribosyl Cyclase ◽  
Sea Urchin Egg ◽  
Sds Page ◽  
Cyclic Adp Ribose ◽  
Charge Analysis

Cyclic ADP-ribose (cADPR) is a metabolite of NAD+ that is as active as inositol trisphosphate (IP3) in mobilizing intracellular Ca2+ in sea urchin eggs. The activity of the enzyme responsible for synthesizing cADPR is found not only in sea urchin eggs but also in various mammalian tissue extracts, suggesting that cADPR may be a general messenger for Ca2+ mobilization in cells. An aqueous soluble enzyme, thought to be an NADase, has been purified recently from the ovotestis of Aplysia californica (Hellmich and Strumwasser, 1991). This paper shows that the Aplysia enzyme catalyzes the conversion of NAD+ to cADPR and nicotinamide. The Aplysia enzyme was purified by fractionating the soluble extract of Aplysia ovotestis on a Spectra/gel CM column. The purified enzyme appeared as a single band of approximately 29,000 Da on SDS-PAGE but could be further separated into multiple peaks by high-resolution, cation-exchange chromatography. All of the protein peaks had enzymatic activity, indicating that the enzyme had multiple forms differing by charge. Analysis of the reaction products of the enzyme by anion-exchange high-pressure liquid chromatography (HPLC) indicated no ADP-ribose was produced; instead, each mole of NAD+ was converted to equimolar of cADPR and nicotinamide. The identification of the product as cADPR was further substantiated by proton NMR and also by its Ca(2+)-mobilizing activity. Addition of the product to sea urchin egg homogenates induced Ca2+ release and desensitized the homogenate to authentic cADPR but not to IP3. Microinjection of the product into sea urchin eggs elicited Ca2+ transients as well as the cortical exocytosis reaction. Therefore, by the criteria of HPLC, NMR, and calcium-mobilizing activity, the product was identical to cADPR. To distinguish the Aplysia enzyme from the conventional NADases that produce ADP-ribose, we propose to name it ADP-ribosyl cyclase.

scholarly journals The role of ATP in the differential ability of Sr2+ to trigger Ca2+ oscillations in mouse and human eggs

2021 ◽  
Author(s):  
Anna Storey ◽  
Khalil Elgmati ◽  
Yisu Wang ◽  
Paul Knaggs ◽  
Karl Swann
Keyword(s):  
Phospholipase C ◽  
Egg Activation ◽  
Apparent Difference ◽  
Free Medium ◽  
Inositol 1 ◽  
Atp Levels ◽  
Mature Mouse ◽  
Differential Ability ◽  
Mouse Eggs

Abstract At fertilization in mice and humans, the activation of the egg is caused by a series of repetitive Ca2+ oscillations which are initiated by phospholipase-C(zeta)ζ that generates inositol-1-4-5-trisphophate (InsP3). Ca2+ oscillations and egg activation can be triggered in mature mouse eggs by incubation in Sr2+ containing medium, but this does not appear to be effective in human eggs. Here we have investigated the reason for this apparent difference using mouse eggs, and human eggs that failed to fertilize after IVF or ICSI. Mouse eggs incubated in Ca2+-free, Sr2+-containing medium immediately underwent Ca2+ oscillations but human eggs consistently failed to undergo Ca2+ oscillations in the same Sr2+ medium. We tested the InsP3-receptor (IP3R) sensitivity directly by photo-release of caged InsP3 and found that mouse eggs were about 10 times more sensitive to InsP3 than human eggs. There were no major differences in the Ca2+ store content between mouse and human eggs. However, we found that the ATP concentration was consistently higher in mouse compared to human eggs. When ATP levels were lowered in mouse eggs by incubation in pyruvate-free medium, Sr2+ failed to cause Ca2+ oscillations. When pyruvate was added back to these eggs, the ATP levels increased and Ca2+ oscillations were induced. This suggests that ATP modulates the ability of Sr2+ to stimulate IP3R-induced Ca2+ release in eggs. We suggest that human eggs may be unresponsive to Sr2+ medium because they have a lower level of cytosolic ATP.

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