A Method for Preparation, Storage, and Activation of Large Populations of Immotile Sea Urchin Sperm

Bracho, Gerácimo E.; Fritch, Jennifer J.; Tash, Joseph S.

doi:10.1006/bbrc.1997.7074

Cited by 18 publications

(17 citation statements)

References 20 publications

(8 reference statements)

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“…It should be noted that cAMP is elevated by 1.9 fold upon dilution from low-pH ASW to Na + -free, Ca 2+ -free ASW added with amine (Beltrán et al, 1996), conditions similar to the present. However, the reactivation conditions allow variable levels of motility to be expressed without cAMP incubation, with some species, including Hemicentrotus, being already at a high percent motility (Murofushi et al, 1986;Brokaw, 1987a;Bracho et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

“…This solution, which will be referred to as KEG, substantially retards motility initiation in sea-urchin sperm cells as described later on. The idea of retardation by KEG is based on the facts that sea-urchin sperm remain quiescent in Na + -free seawater (Lee et al ., 1983;Bibring et al ., 1984;Schackmann et al ., 1984;Gatti and Christen, 1985;Christen et al ., 1986), and also immotile at elevated K + concentrations (Christen et al ., 1986;Bracho et al ., 1997) and become active when the intracellular pH is elevated, e.g., by addition of Na + or amine (Christen et al ., 1982;Lee et al ., 1983;Bibring et al ., 1984;Lee, 1984a, b;Bracho et al ., 1997), and that activation is, however, delayed in the presence of a variety of metal chelators including EDTA (Clapper et al ., 1985;Christen et al ., 1986). The KEG retardation of motility initiation first revealed distinct transient patterns of flagellar bending described in the present study.…”

Section: Introductionmentioning

confidence: 99%

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Progression of Flagellar Stages during Artificially Delayed Motility Initiation in Sea Urchin Sperm

2004

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Progression of Flagellar Stages during Artificially Delayed Motility Initiation in Sea Urchin Sperm

2004

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“…To properly evaluate the role of kinases in the initiation of motility of intact spermatozoa, we first had to find a medium in which these cells could remain completely immotile during a few minutes at 20°C without harmful effects so that the subsequent dilution in seawater would allow motility. We tested two media previously reported to keep sea urchin quiescent (Bracho et al, 1997;Cheung, 1995). These media did not meet the above-mentioned criteria in our hands.…”

Section: Motility Of Intact L Pictus Spermatozoa In the Presence Ofmentioning

confidence: 99%

“…These media did not meet the above-mentioned criteria in our hands. In one case, a low percentage of spermatozoa (Х3 to 5%) initiated motility two minutes after dilution in the medium even though the latter had a pH of 6 and contained 50·mmol·l -1 KCl (Bracho et al, 1997). The other medium was a pH 7.9 Na + -free seawater where choline chloride was substituted for NaCl (Cheung, 1995).…”

Section: Motility Of Intact L Pictus Spermatozoa In the Presence Ofmentioning

confidence: 99%

Protein kinase C is an important signaling mediator associated with motility of intact sea urchin spermatozoa

White

Lamirande

Gagnon

2007

Journal of Experimental Biology

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SUMMARY Numerous kinases and phosphatases are most likely implicated in sperm motility initiation and maintenance. Data on these signaling molecules were mostly obtained from studies conducted on in vitrodemembranated–reactivated sperm models but are not necessarily representative of the in vivo situation. We therefore investigated the effect of a variety of cell-permeable chemicals, mostly kinase inhibitors,on the motility initiation and maintenance of intact sea urchin spermatozoa. Among the 20 substances tested, the protein kinase C (PKC) inhibitor chelerythrine was the most potent, arresting motility at concentrations starting from 1.5–2 μmol l–1. Motility was also inhibited by two other PKC inhibitors as well as staurosporine. Furthermore,these inhibitors prevented the motility-associated increase in phosphorylation of at least four PKC substrates. These phospho-PKC target proteins, as assessed with an antibody specific to phosphorylated motifs of PKC substrates,were found to be associated with the flagellum, either in the Triton X-100 soluble portion or the axoneme (Triton X-100 insoluble). A phosphorylated PKC-like enzyme was also detected by immunoblotting in the flagellum, as well as a significant 50 kDa PKC cleavage product. Taken together, the data strongly indicate for the first time that, in vivo, which means on intact spermatozoa, PKC is a key signaling mediator associated with the maintenance of sea urchin sperm motility.

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“…In previous studies of sperm motility, permeabilization has been accomplished primarily by treating the sperm with non-ionic detergents [1][2][3][4][5][6][7][8][9][10][11][12][13][14]. Although this method has been successfully used to study protein phosphorylation as it relates to motility, detergent treatment has the major disadvantages that membranes, membrane proteins, and many intracellular proteins are solubilized.…”

Section: Introductionmentioning

confidence: 99%

Maintenance of Motility in Mouse Sperm Permeabilized with Streptolysin O1

Johnson

Moss

Gerton

1999

Biology of Reproduction

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One approach to studying the mechanisms governing sperm motility is to permeabilize sperm and examine the regulation of motility by manipulating the intracellular milieu of the cell. The most common method of sperm permeabilization, detergent treatment, has the disadvantage that the membranes and many proteins are extracted from the cell. To avoid this problem, we have developed a method that uses streptolysin O to create stable pores within the plasma membrane while leaving internal membranes intact. Sperm were permeabilized, preincubated, and then treated with 0.6 U/ml of streptolysin O. Permeabilization was assessed by fluorescent dye technologies and endogenous protein phosphorylation using exogenously added [gamma-32P]ATP. Streptolysin O-induced permeabilization rendered the sperm immotile, and the effect was Ca2+-dependent. When the cells were treated simultaneously with a medium containing ATP, streptolysin O-treated sperm maintained flagellar movement. These results demonstrate that the streptolysin O permeabilization model system is a useful experimental method for studying the mechanisms that regulate sperm motility since it allows the flagellar apparatus to be exposed to various exogenously added molecules.

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A Method for Preparation, Storage, and Activation of Large Populations of Immotile Sea Urchin Sperm

Cited by 18 publications

References 20 publications

Progression of Flagellar Stages during Artificially Delayed Motility Initiation in Sea Urchin Sperm

Progression of Flagellar Stages during Artificially Delayed Motility Initiation in Sea Urchin Sperm

Protein kinase C is an important signaling mediator associated with motility of intact sea urchin spermatozoa

Maintenance of Motility in Mouse Sperm Permeabilized with Streptolysin O1

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