Sperm-Surface Chymotrypsin-like Protease Activity Required for Fertilization in Ascidians

Koch, Robert; Norton, Michael L.; Vázquez, Hilda; Lambert, Charles C.

doi:10.1006/dbio.1994.1100

Cited by 24 publications

(12 citation statements)

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“…This fact suggests that vitelline coat lysins are associated with the surface of the plasma membrane of the sperm head, as has been suggested previously (Fukumoto 1983(Fukumoto , 1986(Fukumoto , 1988(Fukumoto , 1990c. This assumption is also supported by the recent findings of a sperm surface chymotrypsin-like enzyme in Ascidia ceratodes, Ascidia callosa, and Ascidia paratropa (Koch et al 1994). Although the chemical nature and the precise role of the acrosomal substance remain to be elucidated, the fact that the acrosome reaction occurs in the perivitelline space and releases a relatively small amount of acrosome substance leads us to the working hypothesis that this substance is responsible for membrane fusion between the apical processes and the egg plasma membrane in ascidians.…”

Section: Acrosome Differentiationsupporting

confidence: 83%

Acrosome differentiation in the ascidians Clavelina lepadiformis and Ciona intestinalis

Fukumoto

2000

Cell and Tissue Research

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The spermatozoa of both Clavelina lepadiformis and Ciona intestinalis have architectural features characteristic of ascidian spermatozoa that have been previously described. They have an elongated head (6 microm and 3 microm long, respectively) and a single mitochondrion that is closely applied laterally to the nucleus; they lack a midpiece. The acrosome of Clavelina lepadiformis spermatozoa is a moderately electron-dense, pear-shaped flattened vesicle, approx. 300 nm x 200 nm x 40 nm in length, width, and height, respectively. The acrosome of Ciona intestinalis spermatozoa is a moderately electron-dense, round flattened vesicle with an electron-dense plate in its central region. It is approx. 200 nm x 200 nm x 50 nm in length, width, and height, respectively. During spermiogenesis in both ascidians, several proacrosomal vesicles (50-70 nm in diameter) appear in a blister at the future apex of the spermatids. These vesicles appear to be associated with the inner surface of the plasma membrane enclosing the blister. They come into contact with each other along the inner surface of the plasma membrane and fuse to form a horseshoe-shaped acrosomal vesicle, which becomes a round, flattened vesicle during further differentiation. Some speculations about the mechanism of acrosome differentiation, the possible role of the acrosome during fertilization, and in the speciation of ascidians are presented.

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Section: Acrosome Differentiationsupporting

confidence: 83%

Acrosome differentiation in the ascidians Clavelina lepadiformis and Ciona intestinalis

Fukumoto

2000

Cell and Tissue Research

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“…The chymotrypsin-like activity of the proteasome was assayed using the cell impermeable, fluorogenic substrate SLLVY-AMC (Koch et al, 1994;Sawada et al, 2002b;Morales et al, 2004). One hundred microliters aliquots of enzyme extracts were incubated in a final volume of 2 ml containing homogenization buffer and 10 mM substrate.…”

Section: Materials and Methods Reagentsmentioning

confidence: 99%

“…Sperm surface labeling. Motile sperm (20 Â 10 6 /ml), selected by swim-up as described above, were resuspended in PBS and incubated with 0.5 mg/ml cellimpermeable NHS-LC-biotin for 30 min at 378C, as described (Koch et al, 1994;Sawada et al, 2002b;Morales et al, 2004). After labeling, the sperm were washed twice with PBS by centrifugation at 300g for 5 min and then the membrane proteins were extracted incubating with 20 mM Triton X-114 for 30 min at 48C.…”

Section: Proteasome Localizationmentioning

confidence: 99%

Role of the sperm proteasome during fertilization and gamete interaction in the mouse

2005

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In this work, we have investigated the role of the sperm proteasome during in vitro fertilization (IVF) and gamete interaction in the mouse. Proteasome activity was measured in extract and intact sperm using a specific substrate. In addition, sperm were treated with specific proteasome inhibitors and evaluated during IVF, binding to the zona pellucida, and progesterone- and zona pellucida-induced acrosome reactions. In other experiments, sperm membrane proteins were obtained resuspending them in Triton X-114, shaking vigorously and let standing by 4 hr. Soluble sperm proteins were partitioned in the aqueous phase and sperm membrane proteins in the detergent phase. In both phases, proteasome activity was measured. Labeling of cell surface sperm proteins was carried out with the cell-impermeable NHS-LC biotin, extracted with Triton X-114, and mixing with avidin-agarose beads. Nonpermeabilized sperm were incubated with an anti-proteasome monoclonal antibody and evaluated by indirect immunofluorescence. The results indicate that sperm extracts as well as intact sperm had proteasome activity; the sperm proteasome was involved in IVF, specifically during sperm-zona pellucida binding and the acrosome reaction; soluble sperm membrane proteins exhibited proteasome activity; biotin experiments indicated the presence of proteasomes on the sperm surface, which was corroborated by indirect immunofluorescence experiments. All these observations indicate that the mouse sperm proteasome participates in the binding to the zona pellucida and the acrosome reaction and that there is a pool of proteasomes located on the sperm head.

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“…We conclude that (1) suramin acts mainly at the VC level, since we never obtained any electrical response on intact eggs which had not been penetrated by sperm while we frequently detected a fertilization current and sperm penetration in naked eggs; and (2) suramin also affects the process of gamete fusion, since while 35% of the naked eggs responded electrically in contact with this drug, 100% of naked eggs inseminated normally in SW displayed a fertilization current. Possibly suramin inhibits one of the sperm proteases involved in ascidian fertilization (De Santis et al, 1992;Koch et al, 1994;Takizawa et al, 1993;Sawada and Someno, 1996); additional experiments are planned to check this possibility.…”

Section: Discussionmentioning

confidence: 99%

Ascidian eggs block polyspermy by two independent mechanisms: One at the egg plasma membrane, the other involving the follicle cells

et al. 1997

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Many ascidians live in clumps and usually release sperm before the eggs. Consequently, eggs are often spawned into dense clouds of sperm. Because fertilization by more than a single sperm is lethal, ascidians have evolved at least two successive blocks to polyspermy: the rapid release of a glycosidase that inhibits sperm binding to the vitelline coat (VC) and a subsequent change in membrane potential that prevents supernumerary sperm-egg fusion. This paper shows that (1) these two blocks can be uncoupled by the use of suramin, and (2) most of the glycosidase appears to be from the follicle cells, which are accessory cells on the outside of the egg VC. Phallusia mammillata eggs initially bind numerous sperm but, after the glycosidase is released, only a few additional sperm bind. Intact eggs in 20 microM suramin release glycosidase, but the electrical response is inhibited; sperm swim actively and bind to the VC but fail to penetrate. Suramin treatment is completely reversible; intact eggs exhibit the electrical response an average of 11 minutes after the drug is washed out. Sperm must contact the follicle cells before passing through the VC; eggs with the VC removed and fertilized in the presence of 20 microM suramin show the electrical response 35% of the time, thus VC removal enhances sperm entry. Like the intact eggs, 100% of the naked eggs respond electrically to fertilization after the drug is washed out. Follicle cells that are isolated by calcium magnesium free seawater and then returned to complete seawater release N-acetylglucosaminidase activity in response to sperm. Thus, these eggs have two blocks to polyspermy that operate in sequence: an early first block resulting from enzymatic modification of the VC by N-acetylglucosaminidase released primarily from follicle cells and a second electrical block operating at the egg plasma membrane level and requiring sperm-egg fusion.

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Sperm-Surface Chymotrypsin-like Protease Activity Required for Fertilization in Ascidians

Cited by 24 publications

References 0 publications

Acrosome differentiation in the ascidians Clavelina lepadiformis and Ciona intestinalis

Acrosome differentiation in the ascidians Clavelina lepadiformis and Ciona intestinalis

Role of the sperm proteasome during fertilization and gamete interaction in the mouse

Ascidian eggs block polyspermy by two independent mechanisms: One at the egg plasma membrane, the other involving the follicle cells

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