Identification of phospholipase activity in Rhinella arenarum sperm extract capable of inducing oocyte activation

Zygote ◽  
2013 ◽  
Vol 22 (4) ◽  
pp. 483-495 ◽  
Author(s):  
Federico Bonilla ◽  
Carlos Minahk ◽  
María Teresa Ajmat ◽  
Graciela Sánchez Toranzo ◽  
Marta Inés Bühler
Keyword(s):  
Ryanodine Receptors ◽  
Cortical Granule ◽  
Oocyte Activation ◽  
Kinase C ◽  
Cyclic Adp Ribose ◽  
Sperm Extract ◽  
Rhinella Arenarum ◽  
Cortical Granule Exocytosis ◽  
Pronuclear Formation

SummaryEgg activation, which includes cortical granule exocytosis, resumption and completion of meiosis and pronuclear formation culminates in the first mitotic cleavage. However, the mechanism through which the fertilizing sperm induces this phenomenon is still controversial. We investigated the effect of the microinjection of homologous sperm soluble fractions obtained by fast protein liquid chromatography (FPLC) from reacted sperm (without acrosome) and non-reacted sperm on the activation of Rhinella arenarum oocytes matured in vitro. The FPLC-purified sperm fraction obtained from reacted or non-reacted sperm is able to induce oocyte activation when it is microinjected. This fraction has a 24 kDa protein and showed phospholipase C (PLC) activity in vitro, which was inhibited by D-609 but not by n-butanol or neomycin, suggesting that it is a PLC that is specific for phosphatidylcholine (PC-PLC). The assays conducted using inhibitors of inositol triphosphate (IP3) and ryanodine receptors (RyRs) indicate that the fraction with biological activity would act mainly through the cADPr (cyclic ADP ribose) pathway. Moreover, protein kinase C (PKC) inhibition blocks the activation produced by the same fraction. Immunocytochemical studies indicate that this PC-PLC can be found throughout the sperm head.

Activation of amphibian oocytes by sperm extracts

Zygote ◽  
2008 ◽  
Vol 16 (4) ◽  
pp. 303-308 ◽  
Author(s):  
F. Bonilla ◽  
M. T. Ajmat ◽  
G. Sánchez Toranzo ◽  
L. Zelarayán ◽  
J. Oterino ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Dna Replication ◽  
Egg Activation ◽  
Cortical Granule ◽  
Oocyte Activation ◽  
Granule Exocytosis ◽  
Bufo Arenarum ◽  
Sperm Extract ◽  
Cortical Granule Exocytosis

SummaryIn the fertilization of most animals, egg activation is accompanied by an increase in cytoplasmatic Ca2+; however, the mechanism through which the fertilizing sperm induce this phenomenon is still controversial. An increase in intracellular free Ca2+ is required to trigger egg activation events, a process that includes cortical granule exocytosis, resumption and completion of meiosis and DNA replication, and culminates in the first mitotic cleavage. In this work, we investigated the effect of microinjection and incubation of different fractions of homologous sperm extract on the activation of Bufo arenarum oocytes matured in vitro. Two heat treatment-sensitive fractions obtained by chromatography were able to induce oocyte activation. The sperm fraction, which contained a 24 kDa protein, induced 90% activation when it was microinjected into the oocytes. Whilst the sperm fraction, which contained a 36 kDa protein, was able to induce about 70% activation only when it was applied on the oocyte surface.

scholarly journals Protein kinase C acts downstream of calcium at entry into the first mitotic interphase of Xenopus laevis.

1990 ◽  
Vol 1 (3) ◽  
pp. 315-326 ◽  
Author(s):  
W M Bement ◽  
D G Capco
Keyword(s):  
Cell Cycle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Xenopus Laevis ◽  
Mitotic Cell ◽  
Cortical Granule ◽  
Mitotic Cell Cycle ◽  
Cleavage Furrow ◽  
Kinase C ◽  
Cortical Granule Exocytosis

Transit into interphase of the first mitotic cell cycle in amphibian eggs is a process referred to as activation and is accompanied by an increase in intracellular free calcium [( Ca2+]i), which may be transduced into cytoplasmic events characteristic of interphase by protein kinase C (PKC). To investigate the respective roles of [Ca2+]i and PKC in Xenopus laevis egg activation, the calcium signal was blocked by microinjection of the calcium chelator BAPTA, or the activity of PKC was blocked by PKC inhibitors sphingosine or H7. Eggs were then challenged for activation by treatment with either calcium ionophore A23187 or the PKC activator PMA. BAPTA prevented cortical contraction, cortical granule exocytosis, and cleavage furrow formation in eggs challenged with A23187 but not with PMA. In contrast, sphingosine and H7 inhibited cortical granule exocytosis, cortical contraction, and cleavage furrow formation in eggs challenged with either A23187 or PMA. Measurement of egg [Ca2+]i with calcium-sensitive electrodes demonstrated that PMA treatment does not increase egg [Ca2+]i in BAPTA-injected eggs. Further, PMA does not increase [Ca2+]i in eggs that have not been injected with BAPTA. These results show that PKC acts downstream of the [Ca2+]i increase to induce cytoplasmic events of the first Xenopus mitotic cell cycle.

The existence of inositol 1,4,5-trisphosphate and ryanodine receptors in mature bovine oocytes

Development ◽  
1995 ◽  
Vol 121 (8) ◽  
pp. 2645-2654 ◽  
Author(s):  
C. Yue ◽  
K.L. White ◽  
W.A. Reed ◽  
T.D. Bunch
Keyword(s):  
Ryanodine Receptors ◽  
Inhibitory Effect ◽  
Ruthenium Red ◽  
Calcium Oscillations ◽  
Ip3 Receptor ◽  
Cyclic Adp Ribose ◽  
Inositol 1,4,5 Trisphosphate ◽  
Bovine Oocytes ◽  
Continuous Injection

Intracellular Ca2+ (Ca2+i) transients during fertilization are critical to the activation of eggs in all species studied. Activation of both the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and ryanodine receptor (RYR) are responsible for the calcium oscillations during fertilization in sea urchin eggs. Using in vitro matured bovine oocytes loaded with Fura-2 AM ester as Ca2+i indicator, we addressed whether IP3Rs and RYRs coexist in mammalian eggs. Our results indicate that microinjection of 50–250 nM IP3 or 10–20 mM caffeine, 100–200 microM ryanodine and 4–8 microM cyclic ADP-ribose all induced Ca2+i release. The Ca2+i release induced by 250 nM IP3 could only be inhibited by prior injection of 1 mg/ml heparin which was overcome by continuous injection of IP3 to 1 microM. Prior injection of either 50 microM ruthenium red, 50 microM procaine or 1 % vehicle medium (VM) did not affect the Ca2+i release induced by IP3. Prior injection of heparin or VM did not affect the Ca2+i release induced by 10–20 mM caffeine or 200 microM ryanodine, but prior injection of 50 microM ruthenium red or procaine completely inhibited the effect of 10–20 mM caffeine. In addition, continuous injection of caffeine up to 40 mM overcame the inhibitory effect of ruthenium red or procaine. The same 50 microM concentration of ruthenium red or procaine only partially blocked the effect of 200 microM ryanodine, but 200 microM ruthenium red or procaine completely blocked the effect of 200 microM ryanodine.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Phosphoprotein inhibition of calcium-stimulated exocytosis in sea urchin eggs.

1991 ◽  
Vol 113 (4) ◽  
pp. 769-778 ◽  
Author(s):  
T Whalley ◽  
I Crossley ◽  
M Whitaker
Keyword(s):  
Sea Urchin ◽  
Okadaic Acid ◽  
Cortical Granule ◽  
Granule Exocytosis ◽  
Sea Urchin Eggs ◽  
Transduction Mechanisms ◽  
Egg Signal ◽  
Cortical Granule Exocytosis ◽  
Gamma S

We have investigated the role of protein phosphorylation in the control of exocytosis in sea urchin eggs by treating eggs with a thio-analogue of ATP. ATP gamma S (adenosine 5'-O-3-thiotriphosphate) is a compound which can be used as a phosphoryl donor by protein kinases, leading to irreversible protein thiophosphorylation (Gratecos, D., and E.H. Fischer. 1974. Biochem. Biophys. Res. Commun. 58:960-967). Microinjection of ATP gamma S inhibits cortical granule exocytosis, but has no effect on the sperm-egg signal transduction mechanisms which normally cause exocytosis by generating an increase in [Ca2+]i. ATP gamma S requires cytosolic factors for its inhibition of cortical granule exocytosis: it does not affect exocytosis when applied directly to the isolated exocytotic apparatus. Our data suggest that ATP gamma S irreversibly inhibits exocytosis via thiophosphorylation of proteins associated with the egg cortex. We have identified two thiophosphorylated proteins (33 and 27 kD) that are associated with the isolated exocytotic apparatus. They may mediate the inhibition of exocytosis by ATP gamma S. In addition, we show that okadaic acid, an inhibitor of phosphoprotein phosphatases, prevents cortical granule exocytosis at fertilization without affecting calcium mobilization. Like ATP gamma S, okadaic acid has no effect on exocytosis in vitro. Our results suggest that an inhibitory phosphoprotein can obstruct calcium-stimulated exocytosis in sea urchin eggs; on the other hand, they do not readily support the idea that a protein phosphatase is an essential component of the mechanism controlling exocytosis.

376 EFFECT OF SPERM TREATMENT ON THE ABILITY TO ACTIVATE OOCYTES AFTER ICSI IN PIGS

2007 ◽  
Vol 19 (1) ◽  
pp. 303
Author(s):  
M. Nakai ◽  
N. Kashiwazaki ◽  
N. Maedomari ◽  
M. Ozawa ◽  
J. Noguchi ◽  
...  
Keyword(s):  
Freeze Drying ◽  
Oocyte Activation ◽  
Sham Injection ◽  
Oocyte Meiosis ◽  
Freeze Dried ◽  
Sperm Penetration ◽  
Sperm Factor ◽  
Pre Treatment ◽  
Pronuclear Formation

During fertilization, sperm penetration (gamete membrane fusion and exposure of sperm cytoplasm) allows oocyte activation (resumption of oocyte meiosis, pronuclear formation, etc.) by inducing an elevation of the intracellular free Ca2+ concentration. So a spermatozoon ought to be able to fully activate an oocyte. However, in pig ICSI oocytes, although a spermatozoon is injected successfully into ooplasm, complete activation is deficient in some of the oocytes. A variety of sperm pre-treatments before ICSI have been reported; however, there is a possibility that the treatment affects the ability to activate oocytes after the injection. We examined the effect of sperm treatments (freezing, freeze-drying, and sonication) on the ability to activate oocytes. Ejaculated boar semen was centrifuged (10 min, 600g) and the supernatant was discarded. The sperm pellet was resuspended in Modena solution (Weitze 1991 Reprod. Domest. Anim. (Suppl. 1), 231–253). The sperm were then treated with or without sonication for 10 s (fresh whole and sonicated sperm, respectively). The freezing of sperm was carried out as was described (Kikuchi et al. 1998 Theriogenology 50, 615–623). Frozen–thawed spermatozoa were then treated with or without sonication (frozen–thawed sonicated and whole sperm, respectively). The fresh whole and sonicated sperm were subjected to a freeze-drying system and the sperm were then re-hydrated (freeze-dried whole and sonicated sperm, respectively). A whole sperm or 1 or 3 sonicated sperm heads were then injected into in vitro-matured oocytes, as described previously (Nakai et al. 2003 Biol. Reprod. 68, 1003–1008; 2006 Reproduction 131, 603–611). Sham injection was also performed. No artificial stimulation was added to the injected oocytes. The oocytes with more than one pronucleus(i) at 10 h after the injection were defined as being activated. As shown in Table 1, the rates of activated oocytes after injection of one sonicated head or sham injection were significantly lower than those of the oocytes injected with whole sperm or 3 sonicated sperm heads in each sperm source (P < 0.05 by ANOVA and Duncan's multiple range test). Furthermore, the rates of activated oocytes for each injection category were not different among the 3 sperm sources. These results suggest that sonication before ICSI may reduce the quantity of activation-inducing sperm factor. It is also suggested that sperm pre-treatment such as freezing or freeze-drying does not affect the ability for oocyte activation. Table 1. Effect of sperm treatment on oocyte activation after ICSI

Participation of PLA2 and PLC in DhL-induced activation of Rhinella arenarum oocytes

Zygote ◽  
2015 ◽  
Vol 24 (4) ◽  
pp. 495-501
Author(s):  
J. Zapata-Martínez ◽  
M.F. Medina ◽  
M.C. Gramajo-Bühler ◽  
G. Sánchez-Toranzo
Keyword(s):  
Aristolochic Acid ◽  
Specific Inhibitor ◽  
Competitive Inhibitor ◽  
Egg Activation ◽  
Inositol Triphosphate ◽  
Phosphoinositide Metabolism ◽  
Oocyte Activation ◽  
Rhinella Arenarum ◽  
Different Doses

SummaryRhinella arenarum oocytes can be artificially activated, a process known as parthenogenesis, by a sesquiterpenic lactone of the guaianolide group, dehydroleucodine (DhL). Transient increases in the concentration of cytosolic Ca2+ are essential to trigger egg activation events. In this sense, the 1-4-5 inositol triphosphate receptors (IP3R) seem to be involved in the Ca2+ transient release induced by DhL in this species. We analyzed the involvement of phosphoinositide metabolism, especially the participation of phospholipase A2 (PLA2) and phospholipase C (PLC) in DhL-induced activation. Different doses of quinacrine, aristolochic acid (ATA) (PLA2 inhibitors) or neomycin, an antibiotic that binds to PIP2, thus preventing its hydrolysis, were used in mature Rhinella arenarum oocytes. In order to assay the participation of PI-PLC and PC- PLC we used U73122, a competitive inhibitor of PI-PLC dependent events and D609, an inhibitor of PC-PLC. We found that PLA2 inhibits quinacrine more effectively than ATA. This difference could be explained by the fact that quinacrine is not a specific inhibitor for PLA2 while ATA is specific for this enzyme. With respect to the participation of PLC, a higher decrease in oocyte activation was detected when cells were exposed to neomycin. Inhibition of PC-PLC with D609 and IP-PLC with U73122 indicated that the last PLC has a significant participation in the effect of DhL-induced activation. Results would indicate that DhL induces activation of in vitro matured oocytes of Rhinella arenarum by activation of IP-PLC, which in turn may induce IP3 formation which produces Ca2+ release.

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