Rapid visual detection of sperm-egg fusion using the DNA-specific fluorochrome Hoechst 33342

Hinkley, Robert E.; Wright, Brent D.; Lynn, John W.

doi:10.1016/0012-1606(86)90082-5

Cited by 101 publications

(55 citation statements)

References 19 publications

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“…The bisbenzimidazole dye H-33342 is a non-toxic DNA-specific stain which allows assessment of DNA organization and distribution within living cells (Arndt-Jovin & Jovin, 1977;Lydon et al, 1980;Cristere'a/., 1983;Hinkley et al, 1986;Luttmer& Longo, 1986). The non-toxic properties of H-33342 depend on cell type and dye concentration, with 1-10 pg H-33342/ml being the typical concentration range used to obtain non-toxic effects in a variety of cell types.…”

Section: Discussionmentioning

confidence: 99%

Pre-loading of mouse oocytes with DNA-specific fluorochrome (Hoechst 33342) permits rapid detection of sperm-oocyte fusion

Conover¹,

Gwatkin²

1988

Reproduction

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Summary. Mouse oocytes exposed to 1 \ g=m\ g Hoechst 33342 (H-33342)/ml and then fertilized in vitro developed normally into blastocysts and blastocyst outgrowths. After penetration of the zona, the fertilizing spermatozoon showed intense fluorescence upon fusion with the vitelline membrane. Due to fluorochrome leakage from the perivitelline space a faint fluorescence was detected in zona-bound spermatozoa. This fluorescence of zona-bound spermatozoa intensified with increased fluorochrome concentration (10\g=m\g/ml),obscuring the fluorescence of the fertilizing spermatozoa. Spermatozoa added to zona-free mouse oocytes (pre-loaded with 1 or 10 \g=m\gH-33342/ml) fluoresced within 10 min of insemination, provided the zonae were removed mechanically. Removal by protease digestion induced leakage of fluorochrome, so that all spermatozoa in the vicinity of an oocyte pre-loaded with 10 pg H-33342/ml became labelled. This leakage was not visibly apparent when protease-treated oocytes were exposed to only 1 \g=m\g H\ x=r eq-\ 33342/ml. The technique could not be applied to zona-free hamster oocytes under our conditions, since the fluorochrome leaked freely from the oocytes whether the zona was removed mechanically or enzymically.

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Section: Discussionmentioning

confidence: 99%

Pre-loading of mouse oocytes with DNA-specific fluorochrome (Hoechst 33342) permits rapid detection of sperm-oocyte fusion

Conover¹,

Gwatkin²

1988

Reproduction

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“…Voltage clamping, either by a whole cell clamp electrode or two intracellular electrodes, is the correct configuration both to "hold" the membrane potential at a particular voltage and to measure the resulting transmembrane currents. Since sea urchin oocytes voltage clamped at negative membrane potentials were shown to be monospermic [50,69], we believe it unlikely that depolarization of the oocyte membrane at activation has evolved to prevent polyspermy.…”

Section: The Fast Polyspermy Block Ideamentioning

confidence: 98%

Polyspermy prevention: facts and artifacts?

Dale¹,

DeFelice

2010

J Assist Reprod Genet

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The purpose of this review is to open a debate as to whether or not oocytes actively repel supernumerary sperm or in nature final sperm : oocyte ratios are so low that polyspermy preventing mechanisms are not necessary. Before encountering the oocyte, spermatozoa need to be primed, either by environmental factors as in animals exhibiting external fertilization, or by factors from the female reproductive tract, as in mammals. The spermatozoon must then recognize and interact with the outer layers of the oocyte and progression of the fertilizing spermatozoon through these layers is further controlled and modulated by a precise sequence of signals in situ. Removal of these outer coats may not inhibit fertilization, however does interfere with the dynamics of sperm-oocyte interaction. We propose that monospermy in mammals and sea urchins, under natural conditions, is ensured by the controlled and gradual encounter of a minimum number of spermatozoa with the oocyte and that fine tuning is ensured by the structural and molecular organization of the oocyte and its surrounding coats. We suggest that laboratory experiments using oocytes deprived of their investments and exposed to unnaturally high concentrations of spermatozoa are artifactual and argue that the conclusions leading to the hypothesis of a fast electrical block to polyspermy are unfounded. Under laboratory conditions the majority of spermatozoa, although motile and capable of attaching to the oocyte surface, are either physiologically incompetent or attach to areas of the oocyte surface that do not support entry.

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“…Using a fluorescent dye to indicate cell-cell fusion, Hinkley et al 21 showed that in sea urchin oocytes voltage clamped at −20 mV, only one spermatozoon fused with the oocyte. Meanwhile, Lynn et al, 22 using clamp voltages of −20 to +17 mV, showed that normal monospermic entry was associated with a typical biphasic fertilization current and quoted "As many as 5-25 sperm may attach before a successful encounter occurs that results in an electrophysiological response."…”

Section: Electrophysiological Data Suggesting a Fast Electrical Blockmentioning

confidence: 99%

“…Sea urchin oocytes can be loaded with a fluorescent dye and cytoplasmic continuity between sperm and oocyte can be determined when the dye flows into the spermatozoon. 21 By inseminating voltage clamped (−20 mV) dye-loaded eggs, Hinkley et al 21 inferred that cell-cell continuity occurred at 4-8 seconds after the initial electrical event.…”

mentioning

confidence: 99%

“…Consequently, there is no reason to believe that activation events would prevent their further progression. The length and density of microvilli, location of cortical granules, 21,27,111 and the priming of the intracellular Ca 2+ release mechanism 112 are events in oocytes that are tightly controlled by the actin cortical cytoskeleton. 113 In the 1970s, it was already suggested that G-actin, sequestered on the membrane, polymerizes during the first few seconds of sperm-oocyte interaction.…”

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confidence: 99%

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Achieving monospermy or preventing polyspermy?

Dale¹

2016

RRB

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Images of sea urchin oocytes with hundreds of spermatozoa attached to their surface have fascinated scientists for over a century and led to the idea that oocytes have evolved mechanisms to allow the penetration of one spermatozoon while repelling supernumerary spermatozoa. Popular texts have extrapolated this concept, to the mammals and amphibians, and in many cases to include all the Phyla. Here, it is argued that laboratory experiments, using sea urchin oocytes deprived of their extracellular coats and inseminated at high densities, are artifactual and that the experiments leading to the idea of a fast block to polyspermy are flawed. Under natural conditions, the number of spermatozoa at the site of fertilization is extremely low, compared with the numbers generated. The sperm:oocyte ratio is regulated first by dilution in externally fertilizing species and the female reproductive tract in those with internal fertilization, followed by a bottleneck created by the oocytes extracellular coats. In order to progress to the oocyte plasma membrane, the fertilizing spermatozoon must encounter and respond to a correct sequence of signals from the oocytes extracellular coats. Those that do not, are halted in their progression by defective signaling and fall to the wayside. Final success and entry is finely tuned by the spermatozoon anchoring to an actin-rich predetermined site on the plasma membrane. In this review, the variation in the form, function, and number of gametes produced across the animal kingdom and the many ways in which sperm-oocyte interactions are regulated to reduce numbers are discussed. Since in nature, final sperm:oocyte ratios approach unity it would appear that selective pressures have favored the achievement of monospermy, rather than the evolution of polyspermy preventing mechanisms.

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Rapid visual detection of sperm-egg fusion using the DNA-specific fluorochrome Hoechst 33342

Cited by 101 publications

References 19 publications

Pre-loading of mouse oocytes with DNA-specific fluorochrome (Hoechst 33342) permits rapid detection of sperm-oocyte fusion

Pre-loading of mouse oocytes with DNA-specific fluorochrome (Hoechst 33342) permits rapid detection of sperm-oocyte fusion

Polyspermy prevention: facts and artifacts?

Achieving monospermy or preventing polyspermy?

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