PSV-11 Efficacy of Quisqualic Acid as an Amino Acid Transporter Inhibitor in Pig Oocytes

2021 ◽  
Vol 99 (Supplement_1) ◽  
pp. 208-208
Author(s):  
Skyla Reynolds ◽  
Haley A Arena ◽  
Kimberly Sprungl ◽  
Brian D Whitaker
Keyword(s):  
Amino Acid ◽  
Cell Expansion ◽  
Cumulus Cell ◽  
Cortical Granule ◽  
Amino Acid Transporters ◽  
Granule Exocytosis ◽  
Quisqualic Acid ◽  
Intracellular Glutathione ◽  
Sodium Dependent ◽  
Cortical Granule Exocytosis

Abstract Quisqualic acid is a known inhibitor of sodium-dependent amino acid transporters. However, it is unknown if quisqualic acid has similar effects in in vitro mature oocytes. Therefore, the objective of this study was to determine the optimal dose and effects of quisqualic acid supplemented during maturation. Oocytes (n=362) were supplemented during maturation with quisqualic acid (0, 0.5, 0.75, 1.0, 2.5 mM) to determine the minimum concentration of quisqualic acid that had no effect on oocyte maturation but significantly decreased the intracellular glutathione concentration. The addition of 1.0 mM quisqualic acid was the lowest concentration observed to cause intracellular glutathione levels to be significantly less (P < 0.05) without affecting maturation compared to no quisqualic acid. Based on those results, oocytes were supplemented with or without 1.0 mM quisqualic acid then evaluated for cumulus cell expansion (n=410) and stage of meiosis (n=380) at the end of maturation. Additional oocytes were fertilized and assessed for cortical granule exocytosis (n=400) and kinetics at 12 h after IVF (n=420). Supplementing quisqualic acid to the media did not have an effect on stage of meiosis, fertilization, polyspermic penetration, or cortical granule exocytosis. Supplementing 1.0 mM quisqualic acid significantly decreased (P < 0.05) cumulus cell expansion by the end of maturation and male pronuclear formation by 12 h after IVF. These results suggest that quisqualic acid supplementation during maturation in pigs inhibits sodium-dependent amino acid transporters.

PSV-10 Effects of l-α-amino Butyrate Supplementation on Oocyte Maturation

2021 ◽  
Vol 99 (Supplement_1) ◽  
pp. 208-209
Author(s):  
Kimberly Sprungl ◽  
Haley A Arena ◽  
Skyla Reynolds ◽  
Brian D Whitaker
Keyword(s):  
Oocyte Maturation ◽  
Cell Expansion ◽  
Cumulus Cell ◽  
Cortical Granule ◽  
Granule Exocytosis ◽  
Beneficial Effects ◽  
Quisqualic Acid ◽  
Surrounding Environment ◽  
Cortical Granule Exocytosis ◽  
Pronuclear Formation

Abstract L-α-amino butyrate is a low-molecular weight thiol compound that acts to increase the levels of glutathione in the oocyte. Glutathione acts as an antioxidant during oocyte maturation and promotes male pronuclear formation during fertilization. Supplementing the L-α-amino butyrate helps to decrease polyspermic penetration rates and improve early embryonic development in swine. However, it is unknown if L-α-amino butyrate supplementation affects the environment of the oocyte or the oocyte directly. Therefore, the objective of this study was to determine if L-α-amino butyrate supplementation to the maturation media acted on the oocyte or had alternative beneficial effects in the surrounding environment. Oocytes were randomly assigned to a maturation media containing an amino acid transport inhibitor, quisqualic acid (QA) (0 or 1 mM) and then supplemented with L-α-amino butyrate (0 or 3.3 mM). Oocytes were evaluated for stage of meiosis (n=380) and cumulus cell expansion (n=411) at the end of maturation. The remaining oocytes were fertilized and evaluated for cortical granule exocytosis (n=400) and IVF kinetics (n=456). Supplementation of L-α-amino butyrate with or without QA significantly increased (P < 0.05) cumulus cell expansion, cortical granule exocytosis and male pronuclear formation compared to no supplementation or QA supplementation. There was no difference in meiotic progression, fertilization or polyspermic penetration rates between the treatment groups. Results suggest that when L-α-amino butyrate is supplemented during maturation, it improves the maturation of the oocyte by acting directly on the oocyte and not through the surrounding environment of the oocyte.

scholarly journals Compensatory endocytosis occurs after cortical granule exocytosis in mouse eggs

2019 ◽  
Vol 235 (5) ◽  
pp. 4351-4360
Author(s):  
Matías D. Gómez‐Elías ◽  
Rafael A. Fissore ◽  
Patricia S. Cuasnicú ◽  
Débora J. Cohen
Keyword(s):  
Cortical Granule ◽  
Granule Exocytosis ◽  
Cortical Granule Exocytosis ◽  
Mouse Eggs

scholarly journals Cortical Granule Exocytosis Is Mediated by Alpha-SNAP and N-Ethilmaleimide Sensitive Factor in Mouse Oocytes

PLoS ONE ◽  
2015 ◽  
Vol 10 (8) ◽  
pp. e0135679 ◽  
Author(s):  
Matilde de Paola ◽  
Oscar Daniel Bello ◽  
Marcela Alejandra Michaut
Keyword(s):  
Cortical Granule ◽  
Granule Exocytosis ◽  
Mouse Oocytes ◽  
Sensitive Factor ◽  
Cortical Granule Exocytosis

scholarly journals Interaction of Excitatory Amino Acid Transporters 1 – 3 (EAAT1, EAAT2, EAAT3) with N-Carbamoylglutamate and N-Acetylglutamate

2017 ◽  
Vol 43 (5) ◽  
pp. 1907-1916 ◽  
Author(s):  
Birgitta C. Burckhardt ◽  
Gerhard Burckhardt
Keyword(s):  
Amino Acid ◽  
Excitatory Amino Acid ◽  
Hepatic Uptake ◽  
Xenopus Laevis Oocytes ◽  
Amino Acid Transporters ◽  
Synthetic Analog ◽  
Excitatory Amino Acid Transporters ◽  
Sodium Dependent ◽  
Inward Currents ◽  
Acetylglutamate Synthase

Background/Aims: Inborn deficiency of the N-acetylglutamate synthase (NAGS) impairs the urea cycle and causes neurotoxic hyperammonemia. Oral administration of N-carbamoylglutamate (NCG), a synthetic analog of N-acetylglutamate (NAG), successfully decreases plasma ammonia levels in the affected children. Due to structural similarities to glutamate, NCG may be absorbed in the intestine and taken up into the liver by excitatory amino acid transporters (EAATs). Methods: Using Xenopus laevis oocytes expressing either human EAAT1, 2, or 3, or human sodium-dependent dicarboxylate transporter 3 (NaDC3), transport-associated currents of NAG, NCG, and related dicarboxylates were assayed. Results: L-aspartate and L-glutamate produced saturable inward currents with Km values below 30 µM. Whereas NCG induced a small inward current only in EAAT3 expressing oocytes, NAG was accepted by all EAATs. With EAAT3, the NAG-induced current was sodium-dependent and saturable (Km 409 µM). Oxaloacetate was found as an additional substrate of EAAT3. In NaDC3-expressing oocytes, all dicarboxylates induced much larger inward currents than did L-aspartate and L-glutamate. Conclusion: EAAT3 may contribute to intestinal absorption and hepatic uptake of NCG. With respect to transport of amino acids and dicarboxylates, EAAT3 and NaDC3 can complement each other.

scholarly journals Poisson-distributed active fusion complexes underlie the control of the rate and extent of exocytosis by calcium.

1996 ◽  
Vol 134 (2) ◽  
pp. 329-338 ◽  
Author(s):  
S S Vogel ◽  
P S Blank ◽  
J Zimmerberg
Keyword(s):  
Poisson Process ◽  
Sea Urchin ◽  
Physiological Responses ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Granule Exocytosis ◽  
Calcium Dependence ◽  
Sea Urchin Egg ◽  
High Calcium ◽  
Cortical Granule Exocytosis

We have investigated the consequences of having multiple fusion complexes on exocytotic granules, and have identified a new principle for interpreting the calcium dependence of calcium-triggered exocytosis. Strikingly different physiological responses to calcium are expected when active fusion complexes are distributed between granules in a deterministic or probabilistic manner. We have modeled these differences, and compared them with the calcium dependence of sea urchin egg cortical granule exocytosis. From the calcium dependence of cortical granule exocytosis, and from the exposure time and concentration dependence of N-ethylmaleimide inhibition, we determined that cortical granules do have spare active fusion complexes that are randomly distributed as a Poisson process among the population of granules. At high calcium concentrations, docking sites have on average nine active fusion complexes.

Weak bases partially activate Xenopus eggs and permit changes in membrane conductance whilst inhibiting cortical granule exocytosis

1987 ◽  
Vol 87 (2) ◽  
pp. 205-220
Author(s):  
M. Charbonneau ◽  
D.J. Webb
Keyword(s):  
Polar Body ◽  
Membrane Conductance ◽  
Transient Increase ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Weak Base ◽  
Granule Exocytosis ◽  
Weak Bases ◽  
Prolonged Contact ◽  
Cortical Granule Exocytosis

At extracellular pH values close to their pKa values the weak bases, ammonia and procaine, elicited a series of events in non-activated Xenopus eggs, some of which resembled those normally occurring at fertilization. These included: (1) a transient increase in membrane conductance; (2) modification of the microvilli; (3) thickening of the cortical cytoplasm and displacement of the cortical granules; (4) pigment accumulation; (5) contractions and shape changes. However, these eggs did not undergo the cortical reaction nor emit the second polar body. Cortical granule exocytosis of inseminated or artificially stimulated eggs was inhibited if the eggs had been previously treated for 15 min with the weak base and subsequently rinsed. Multiple sperm-entry sites were exhibited by the inseminated eggs, suggesting polyspermy. However, such eggs did not cleave and although sperm had fused with the egg membrane, they were not incorporated. Nevertheless, a transient increase in membrane conductance was evoked, which was longer in duration and had a slightly different form from that normally accompanying fertilization. In these eggs cortical granules were intact but displaced away from the plasma membrane. Prolonged contact with the weak base rendered eggs totally unresponsive to sperm or artificial stimuli but eggs recovered when rinsed sufficiently. These effects of weak bases on unfertilized Xenopus eggs or during fertilization were completely absent at pH 7.4. Although changes in intracellular pH or Ca2+ may be involved in these phenomena, direct action by the weak base itself cannot be ruled out.

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.

SNAP-25 is essential for cortical granule exocytosis in mouse eggs

1998 ◽  
Vol 274 (6) ◽  
pp. C1496-C1500 ◽  
Author(s):  
Yoshihide Ikebuchi ◽  
Nobuyuki Masumoto ◽  
Tetsu Matsuoka ◽  
Takeshi Yokoi ◽  
Masahiro Tahara ◽  
...  
Keyword(s):  
Botulinum Neurotoxin ◽  
Endocrine Cells ◽  
Immunoblot Analysis ◽  
Cortical Granule ◽  
Rt Pcr ◽  
Botulinum Neurotoxin A ◽  
Granule Exocytosis ◽  
Cortical Granule Exocytosis ◽  
And Function ◽  
Mouse Eggs

Synaptosome-associated protein of 25 kDa (SNAP-25) has been shown to play an important role in Ca2+-dependent exocytosis in neurons and endocrine cells. During fertilization, sperm-egg fusion induces cytosolic Ca2+mobilization and subsequently Ca2+-dependent cortical granule (CG) exocytosis in eggs. However, it is not yet clear whether SNAP-25 is involved in this process. In this study, we determined the expression and function of SNAP-25 in mouse eggs. mRNA and SNAP-25 were detected in metaphase II (MII) mouse eggs by RT-PCR and immunoblot analysis, respectively. Next, to determine the function of SNAP-25, we evaluated the change in CG exocytosis with a membrane dye, tetramethylammonium-1,6-diphenyl-1,3,5-hexatriene, after microinjection of a botulinum neurotoxin A (BoNT/A), which selectively cleaves SNAP-25 in MII eggs. Sperm-induced CG exocytosis was significantly inhibited in the BoNT/A-treated eggs. The inhibition was attenuated by coinjection of SNAP-25. These results suggest that SNAP-25 may be involved in Ca2+-dependent CG exocytosis during fertilization in mouse eggs.

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