Determining acrosomal status of the cynomolgus monkey (<i>Macaca fascicularis</i>) sperm by fluorescence microscopy

Cross, Nicholas L.; Morales, Patricio; Fukuda, Masaru; Behboodi, E.

doi:10.1002/ajp.1350170205

Cited by 23 publications

(6 citation statements)

References 10 publications

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“…With this aim, in vitro capacitated sperm were exposed for 30 minutes to solubilized ZP and the resulting total acrosome reaction (AR) was detected by FITC-PSA staining (Ward and Storey, 1984;Barboni et al, 1995). PSA has been shown to bind selectively to the acrosomal content on the sperm head in a number of species [human (Cross et al, 1986); monkey (Cross et al, 1989); pig (Barboni et al, 1995)]. Intense staining of the acrosomal content, with much less label on the other regions of the cell, is indicative of an intact acrosome.…”

Section: Detection Of Sperm Capacitationmentioning

confidence: 99%

Characterization of the endocannabinoid system in boar spermatozoa and implications for sperm capacitation and acrosome reaction

Maccarrone

Barboni

Paradisi

et al. 2005

Journal of Cell Science

187

234

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Anandamide (AEA) is the endogenous ligand of cannabinoid (CB) receptors, and as such it plays several central and peripheral activities. Regulation of female fertility by AEA has attracted growing interest, yet a role for this endocannabinoid in controlling sperm function and male fertility in mammals has been scarcely investigated. In this study we report unprecedented evidence that boar sperm cells have the biochemical machinery to bind and degrade AEA, i.e. type-1 cannabinoid receptors (CB1R), vanilloid receptors (TRPV1), AEA-synthesizing phospholipase D (NAPE-PLD), AEA transporter (AMT) and AEA hydrolase (FAAH). We also show that the non-hydrolyzable AEA analogue methanandamide reduces sperm capacitation and, as a consequence, inhibits the process of acrosome reaction (AR) triggered by the zona pellucida, according to a cyclic AMP-dependent pathway triggered by CB1R activation. Furthermore, activation of TRPV1 receptors seems to play a role of stabilization of the plasma membranes in capacitated sperm, as demonstrated by the high incidence of spontaneous AR occurring during the cultural period when TRPV1 activity was antagonized by capsazepine. We show that sperm cells have a complete and efficient endocannabinoid system, and that activation of cannabinoid or vanilloid receptors controls, at different time-points, sperm functions required for fertilization. These observations open new perspectives on the understanding and treatment of male fertility problems[...

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Section: Detection Of Sperm Capacitationmentioning

confidence: 99%

Characterization of the endocannabinoid system in boar spermatozoa and implications for sperm capacitation and acrosome reaction

Maccarrone

Barboni

Paradisi

et al. 2005

Journal of Cell Science

187

234

View full text Add to dashboard Cite

show abstract

“…Sperm were assessed simultaneously for viability and acrosomal status using the supravital dye Hoechst 33258 (H258) and fluorescein isothionate-conjugated Pisum sativum agglutinin (FITC-PSA; Vector Laboratories, Burlingame, CA) [16,17]. The method of Morales and Cross [18] for acrosomal staining of human spermatozoa on small-pore polycarbonate filters as modified by Tollner et al [14] for application to macaque spermatozoa was used.…”

Section: Assessment Of Sperm Motility and Acrosomal Statusmentioning

confidence: 99%

“…After 1 min or 4 min of coincubation, the oocytes were quickly rinsed in the same incubation medium and immediately fixed in cold absolute ethanol for 1-2 min. After fixation, oocytes were placed on glass slides, and acrosomal status of sperm bound to the zonae was determined by indirect immunofluorescence using a polyclonal antisperm antiserum produced and employed as described by Cross et al [16,17]. The labeling technique of Cross et al [16] was modified by performing the labeling directly on the glass slide rather than transferring oocytes through drops of the various solutions [11].…”

Section: Assessment Of Sperm-zona Binding and Acrosomal Status On Thementioning

confidence: 99%

Interaction of Acrosome-Reacted Macaque Sperm with the Macaque Zona Pellucida1

VandeVoort¹,

Yudin²,

Overstreet³

1997

Biology of Reproduction

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In this study we investigated the ultrastructure of macaque sperm induced to acrosome-react with calcium ionophore A23187, and the interaction between these acrosome-reacted sperm and the macaque zona pellucida. Transmission electron microscopy revealed that the majority of ionophore-treated sperm retained the vesiculated acrosomal cap or "shroud." Untreated, acrosome-reacted sperm on the zona had a similar ultrastructural appearance. In sperm-zona binding experiments, a mean of 4.5 ionophore-treated sperm were bound per zona after 1 min of coincubation compared with 41 sperm per zona in the solvent control. Vigorous pipetting was used to remove the acrosomal shrouds from approximately 50% of acrosome-reacted sperm before incubation with oocytes. Significantly more of these mechanically treated sperm were bound to the zona after a 4-min coincubation compared with acrosome-reacted sperm that were not pipetted. The number of mechanically treated sperm bound to the zona was the same whether the sperm and oocytes were coincubated in calcium-free medium or in control medium. The percentage of mechanically treated sperm that were acrosome-reacted on the zona also was not different in the two media. We conclude that macaque sperm that undergo the acrosome reaction on the zona surface are bound by the acrosomal shroud before zona penetration. When sperm acrosome-react before interaction with the oocyte, their zona binding capacity is significantly reduced. Removal of the acrosomal shroud and exposure of the inner acrosomal membrane increases the affinity of sperm for the zona. This sequence occurs naturally during the transition from primary binding to secondary binding on the zona surface.

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“…The remaining oocytes were placed into 100 p1 drops of fresh BWW without sperm under oil and incubated for 1 hr ("chase") before fixation in ethanol. After fixation, oocytes were placed on glass slides and acrosomal status of sperm bound to the zonae was determined by indirect immunofluorescence using a polyclonal antisperm antiserum produced and employed as described by (Cross et al, 1986(Cross et al, ,1989. The labelling technique of Cross et al (1986) was modified by performing the labelling directly on the glass slide rather than transferring oocytes through drops of the various solutions.…”

Section: Zona Binding Experiments Andmentioning

confidence: 99%

Separate effects of caffeine and dbcAMP on macaque sperm motility and interaction with the zona pellucida

VandeVoort

Tollner

Overstreet

1994

Molecular Reproduction Devel

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Capacitation of macaque sperm with caffeine and dbcAMP is required for fertilization in vitro. This study determined the separate effects of caffeine and dbcAMP on sperm-zona pellucida binding and the acrosome reaction of zona bound sperm. Semen from 6 cynomolgus macaques was washed through 60% Percoll, resuspended, and washed with BWW media and incubated for 2.5 hr. Caffeine, dbcAMP (2 mM each), or both (1 mM each) were added to aliquots of the sperm suspensions. Immature macaque oocytes were placed into drops of sperm suspensions, coincubated with sperm for 30 sec, and either fixed immediately or removed to sperm-free media and incubated 1 hr before fixation. There were no significant differences between groups in the percentage of live, acrosome-reacted sperm in suspension. Treatment with caffeine and dbcAMP or with caffeine alone, significantly increased the number of sperm bound to each zona pellucida (96 +/- 16 and 81 +/- 17, respectively) compared to control and dbcAMP treatment (15 +/- 4 and 28 +/- 13). However, treatment with dbcAMP, alone and with caffeine, resulted in a higher percentage of acrosome-reacted sperm on the zona (15.2 +/- 2.1 and 9.0 +/- 0.6) than control or caffeine treatment (3.0 +/- 1.4 and 2.4 +/- 0.5). Effects on sperm motility consistent with hyperactivation were detected only when both caffeine and dbcAMP were present. Although both caffeine and dbcAMP are presumed to increase or to produce the same effect as increased intracellular cAMP levels, these compounds have different effects on the ability of sperm to bind to the zona and to undergo the acrosome reaction.

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Determining acrosomal status of the cynomolgus monkey (Macaca fascicularis) sperm by fluorescence microscopy

Cited by 23 publications

References 10 publications

Characterization of the endocannabinoid system in boar spermatozoa and implications for sperm capacitation and acrosome reaction

Characterization of the endocannabinoid system in boar spermatozoa and implications for sperm capacitation and acrosome reaction

Interaction of Acrosome-Reacted Macaque Sperm with the Macaque Zona Pellucida1

Separate effects of caffeine and dbcAMP on macaque sperm motility and interaction with the zona pellucida

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