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Biomedical subjects

N L Cross

Publications and source records attributed to N L Cross.

At least 19 recordsLinked to original sources
PubMed2000

Sphingomyelin modulates capacitation of human sperm in vitro.

Ejaculated mammalian sperm must mature (capacitate) before they can undergo acrosomal exocytosis and fertilize an egg. Loss of sperm sterols is an early step in capacitation. Because sphingomyelin slows cholesterol efflux from other cells, the role of sphingomyelin in capacitation was tested. Human sperm were exposed to sphingomyelinase and then incubated for as long as 24 h. The ability of sperm to acrosome-react in response to progesterone was tested to measure capacitation. Sphingomyelinase-treated sperm became responsive to progesterone approximately 10 h earlier than control sperm. Sphingomyelinase also increased spontaneous acrosomal exocytosis. The effects of sphingomyelinase were accompanied by accelerated losses of the inhibitory sterols, cholesterol and desmosterol. To test whether sphingomyelinase-generated ceramide might promote capacitation, sperm were incubated for 8 h with the cell-permeable ceramide N:-hexanoylsphingosine (25 microM) or with solvent. Ceramide increased the incidence of progesterone-responsive sperm and, at later times, spontaneously reacted sperm. N:-Hexanoylsphinganine, an inactive control ceramide, had no effect. These results suggest that sphingomyelin in the sperm influences the rate of capacitation by slowing the loss of sterols, and that exogenous sphingomyelinase accelerates capacitation by speeding the loss of sterols and by generating ceramide.

Acrosome Reaction↗
PubMed1999

Effect of methyl-beta-cyclodextrin on the acrosomal responsiveness of human sperm.

Human sperm incubated in vitro gradually become capable of acrosome-reacting in response to the agonist, progesterone (P4). Loss of unesterified cholesterol is an obligatory step in the development of acrosomal responsiveness. These experiments tested the ability of methyl-beta-cyclodextrin (MbetaCD) to accelerate sperm cholesterol loss and the development of acrosomal responsiveness. Incubating sperm 30 min in MbetaCD (2.5-10 mM) decreased sperm cholesterol by as much as 89% in a dose-dependent fashion. MbetaCD caused some sperm (maximum of 16% following treatment with 5 mM MbetaCD) to become responsive to P4, and it caused a dose-dependent increase in spontaneous acrosome reactions. The number of responsive sperm increased in the first 3 hr following their removal from MbetaCD. Continuing incubation to 24 hr increased the numbers of spontaneously reacted sperm and dead sperm, but not P4-responsive sperm. It appears, therefore, that up to 3 hr are required for the full expression of P4-responsiveness in cholesterol-depleted sperm. The observed effects of MbetaCD are due to its cholesterol-depleting properties, because including sufficient cholesterol with MbetaCD to reduce the loss of sperm cholesterol inhibited the effects of MbetaCD on cell viability, spontaneous acrosome reactions, and responsiveness to P4. MbetaCD accelerates the appearance of the functional stages that sperm normally pass through during incubation in vitro, reinforcing the view that cholesterol loss is an important determinant of the rate at which sperm become acrosomally responsive.

Acrosome↗
PubMed1997

Control of human sperm intracellular pH by cholesterol and its relationship to the response of the acrosome to progesterone.

When incubated in vitro, human sperm gradually become capable of acrosome-reacting in response to the agonist progesterone. Loss of unesterified cholesterol is required for sperm to become responsive to progesterone, but how cholesterol regulates acrosomal responsiveness is unknown. These experiments tested the hypothesis that loss of sperm cholesterol leads to a rise in the intracellular pH (pH(i)) that makes the sperm responsive to progesterone. pH(i) was measured using BCECF (2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein) in freshly ejaculated sperm (T0 sperm) and in sperm incubated in vitro overnight (T24 sperm). During incubation, pH(i) increased from 6.94 +/- 0.03 to 7.08 +/- 0.01 (mean +/- SEM, n = 4, p < 0.01). Incubating sperm 24 h in medium supplemented with 1 microM cholesterol to prevent loss of sperm cholesterol suppressed the rise of pH(i) (T24C sperm, pH(i) = 6.96 +/- 0.03, n = 4, p = 0.64 compared to T0 sperm). To test whether their lower pH(i) prevents T24C sperm from reacting, we treated T24C sperm with the alkalinizing agents trimethylamine chloride (TMA) or NH4Cl. These agents did cause T24C sperm to respond to progesterone in a dose-dependent fashion, but they also caused a similar increase in the number of reacting T24 sperm. These agents probably do not reverse the inhibiting effects of high cholesterol but rather make responsive a subpopulation of sperm that is present regardless of the cholesterol content. NH4Cl and TMA did not make T0 sperm responsive to progesterone. The acidifying agent sodium propionate did not diminish the response of T24 sperm to progesterone. In summary, pH(i) increases during incubation in vitro in a cholesterol-dependent fashion. Elevated pH(i) alone is probably not sufficient to make sperm acrosomally responsive.

Acrosome↗
PubMed1996

Effect of cholesterol and other sterols on human sperm acrosomal responsiveness.

Human sperm become responsive to inducers of the acrosome reaction when they are washed free of seminal plasma and incubated in an appropriate medium. Previous work has shown that cholesterol-enriched medium prevents sperm from becoming responsive to the inducer, progesterone. Sperm that were incubated 24 hr in cholesterol-enriched medium and then treated with progesterone showed no evidence of membrane fusion, indicating that cholesterol acts at a stage before the earliest morphological change. Inhibition of acrosomal responsiveness by cholesterol was reversible. Among other sterols reported in mammalian sperm, desmosterol and cholesterol sulfate also inhibited sperm from becoming responsive, but cholesterol palmitate did not. Our results support a model in which sperm unesterified cholesterol, or a molecule in equilibrium with it, suppresses acrosomal responsiveness. Cholesterol-enriched medium also prevented sperm from becoming responsive to the calcium/proton exchanging ionophore, ionomycin, suggesting that cholesterol's effect may be, at least in part, at a point in the signal transduction pathway subsequent to the rise in intracellular-free calcium.

Acrosome↗
PubMed1996

Human seminal plasma prevents sperm from becoming acrosomally responsive to the agonist, progesterone: cholesterol is the major inhibitor.

Seminal plasma inhibits human sperm from developing the ability to undergo the acrosome reaction. The inhibitory activity was identified as that of cholesterol on the basis of its solubility in organic solvents, its chromatographic behavior (adsorption, thin-layer, and gas chromatography), and its mass spectrum. Contrary to findings in other reports, no evidence for inhibitory proteins or peptides was found, and spermine was not an effective inhibitor. The inhibitory activity of untreated seminal plasma from individual ejaculates was highly correlated with the cholesterol content of the ejaculates (r = 0.96), suggesting that the amount of cholesterol determines the inhibitory activity of unfractionated seminal plasma. The inhibitory activity of unfractionated seminal plasma was significantly less, relative to the cholesterol content, than the activity of pure cholesterol, which is consistent with the idea that there are components in seminal plasma that partially counter the effect of cholesterol by promoting the development of acrosomal responsiveness.

Acrosome↗
PubMed1996

Unesterified cholesterol content of human sperm regulates the response of the acrosome to the agonist, progesterone.

Human sperm become responsive to inducers of the acrosome reaction if they are washed free of seminal plasma and incubated in an appropriate medium. We tested the hypothesis that sperm must lose cholesterol during incubation in order to become responsive to the agonist, progesterone. Freshly ejaculated sperm contained 2.92 +/- 0.202 nmol unesterified cholesterol/10(7) sperm (mean +/- SEM, n = 18). When incubated for 24 h in vitro, sperm suspensions lost 29 +/- 6% of their free cholesterol (n = 23). Sperm lost cholesterol slightly faster than they became acrosomally responsive. Adding cholesterol to the medium prevented sperm from losing cholesterol and from becoming responsive. Varying the cholesterol content of the medium had similar effects on loss of sperm cholesterol (ED50 = 406 nM) and acrosomal responsiveness (ED50 = 388 nM). Incubating sperm with a 1:150 dilution of seminal plasma (containing 5.18 microM cholesterol) also prevented sperm from losing cholesterol and from becoming responsive. Incubating sperm 24 h in medium containing 0.5 mg/ml phosphatidylcholine increased the amount of cholesterol lost and the number of sperm that became responsive. Our results support a model in which sperm unesterified cholesterol (or a molecule in equilibrium with it) suppresses acrosomal responsiveness. Sperm must lose unesterified cholesterol to become responsive to progesterone.

Acrosome↗
PubMed1994

Assessing acrosomal status of bovine sperm using fluoresceinated lectins.

Binding of 12 lectins to bull sperm was analyzed to select a lectin that bound preferentially to the acrosomal region. Peanut agglutinin (PNA) and Pisum sativum agglutinin (PSA) were suitably specific for intracellular, acrosome-associated glycoconjugates. Peanut agglutinin exhibited almost no detectable binding to sperm surface receptors, but intense binding to the area of the acrosome anterior to the equatorial segment. In contrast, PSA bound intensely to anterior and equatorial acrosomal regions, and weakly to the other regions of the sperm. Acrosomal labeling by both lectins decreased when sperm were induced to acrosome-react with calcium ionophore. To determine if these lectins could be used to assess acrosomal status, we compared the percentage of acrosome-reacted sperm that were detected by staining with naphthol yellow and erythrosin B with the percentage that were detected by lectin labeling. The incidence of reacted sperm detected by PSA labeling was not significantly different from that detected by naphthol yellow/ erythrosin B (P = 0.46). The incidence of reacted sperm detected by PNA was correlated with the incidence detected by naphthol yellow/erythrosin B, but was significantly lower (P = 0.003). We conclude that labeling permeabilized sperm with fluoresceinated PSA can serve as a rapid assay for acrosomal status.

Journal Article↗
PubMed1993

Multiple effects of seminal plasma on the acrosome reaction of human sperm.

Mammalian sperm do not respond to inducers of the acrosome reaction immediately after ejaculation. They become responsive after they are removed from seminal plasma and incubated in an appropriate medium. We tested the effects of seminal plasma on the development of acrosomal responsiveness. Washed human sperm incubated 24 hr in vitro with 10% (v/v) seminal plasma did not complete an acrosome reaction when exposed to human follicular fluid, progesterone, or ionomycin. Seminal plasma did not reduce sperm viability or motility. Electron microscopy of sperm incubated 24 hr with 5% seminal plasma and then treated with progesterone revealed no sign of membrane fusion or other changes that are associated with the acrosome reaction. During a 12-hr incubation, seminal plasma was 50% effective at inhibiting the acrosomal response to progesterone when diluted 821 +/- 112 fold (mean +/- SD, n = 3). Sperm that were incubated with seminal plasma for 24 hr and then washed free of the seminal plasma became acrosomally responsive over the following 24 hr, at a rate similar to that of sperm not incubated with seminal plasma in vitro. When sperm were incubated 6 hr without seminal plasma and then seminal plasma was added, the sperm population transiently became more responsive to progesterone, and then became unresponsive. During incubation in vitro, the ability of sperm to have an augmented response to a mixture of seminal plasma plus progesterone developed slightly earlier and more rapidly than ability to respond to progesterone alone. When sperm were incubated 24 hr without seminal plasma, a few acrosome reacted in response to the addition of seminal plasma alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrosome↗
PubMed1991

Effects of cryopreservation on human sperm acrosomes.

Total acrosin activity and acrosomal status were determined before and after cryopreserving human spermatozoa. Three different cryopreservation protocols were used. Both acrosin activity and the incidence of intact acrosomes decreased during cryopreservation. The magnitudes of the decreases were weakly but significantly correlated (r = 0.29, P less than 0.05), suggesting that acrosomal loss contributed to the decrease in acrosin activity. The effects of the three cryopreservation protocols were not significantly different. Motility decreased more (average 43%) than did the percentage of spermatozoa with intact acrosomes (27%) and the total acrosin activity (24%). These measurements suggested that acrosomal damage may have been secondary to cell death. This hypothesis was tested by determining the acrosomal status of spermatozoa that survived cryopreservation. Spermatozoa that were motile after thawing averaged 96% acrosome-intact; their acrosin activity, however, was significantly less than that of motile, unfrozen spermatozoa. These observations support the idea that the acrosomal loss due to cryopreservation is associated with cell death but also demonstrate decreased total acrosin activity of the acrosome-intact spermatozoa that survive cryopreservation.

Acrosin↗
PubMed1990

Regional binding of human anti-sperm antibodies assessed by indirect immunofluorescence.

Indirect immunofluorescence (IIF) can be a powerful tool for determining the site on spermatozoa to which antibodies bind. Human sera that contain anti-sperm antibodies are often of low titre, and may contain antibodies directed against both intracellular and surface antigens. We have developed an IIF protocol that helps to distinguish intracellular from surface labelling. The two types of labelling were differentiated by exposing the spermatozoa to Hoechst 33258, a nuclear stain of low membrane permeability, to tag the spermatozoa that had disrupted membranes. Surface labelling detected in this fashion was patchy. It was much more uniform if the spermatozoa were fixed in paraformaldehyde, or if a univalent, Fab fragment was used as the second antibody. Thus, it is likely that most of the patchy appearance is due to the bivalent second antibody cross-linking mobile antigen-antibody complexes. For some sera, patching was so pronounced that it appeared to remove the label from portions of the sperm surface, giving a misleading picture of the regions to which the antibodies were directed. Fourteen sera were used in IIF and none of them labelled spermatozoa solely on the head or on the tail.

Antibodies↗
PubMed1989

Acrosome intact and acrosome-reacted human sperm can initiate binding to the zona pellucida.

Mammalian sperm must be acrosome reacted before penetrating the zona pellucida. In some species the sperm undergo the acrosome reaction before binding to the zona pellucida and in other species only acrosome intact sperm can initiate binding to the zona. In this study we addressed the question of acrosomal status and sperm-zona binding with human gametes. Sperm acrosome reactions were induced by treatment with human follicular fluid or N-(6-amino-hexyl)-5-chloro-naphthalene sulfonamide (W-7). The sperm suspensions, containing various percentages of acrosome-reacted sperm, were then incubated with human oocytes for 1 min. The acrosomal status of the sperm population bound to the zona was similar to the acrosomal status of the population of sperm in suspension (R2 = 0.77), regardless of the treatment to induce acrosome reactions. Our interpretation of these results is that both acrosome intact and acrosome-reacted human sperm can initiate binding to the zona pellucida. However, we reported earlier (N. L. Cross, P. Morales, J. W. Overstreet, and F. W. Hanson, 1988, Biol. Reprod. 38, 235-244) that the human zona pellucida is able to induce acrosome reactions. Thus, to exclude the possibility that sperm had undergone the acrosome reaction on the zona within 1 min of binding, sperm were suspended in a nominally calcium-free Tyrode's medium (0 Ca-mTyr) before incubation with oocytes (this medium was supplemented with SrCl2 and spermine to support sperm motility and zona binding). In 0 Ca-mTyr, the proportion of acrosome-reacted sperm on the zona was still highly correlated with the proportion of reacted sperm in suspension, indicating that the sperm were reacted before binding. Evidence that 0 Ca-mTyr effectively inhibited acrosome reactions induced by the zona pellucida was derived from experiments in which sperm were treated with human follicular fluid or control medium and the suspensions were diluted with either 0 Ca-mTyr or control medium.4+ Human oocytes were added for 1 min (pulse) at which time some oocytes were fixed and other oocytes were transferred to sperm-free medium and incubated for 35 min (chase) before fixation. Sperm diluted in control medium, pretreated with either human follicular fluid or control medium, showed a similar increase (40%) in the percentage of acrosome reactions among the zona-bound sperm after the chase. Sperm diluted in 0 Ca-mTyr did not show an increase in the percentage of acrosome-reacted sperm on the zona pellucida after the chase.(ABSTRACT TRUNCATED AT 400 WORDS)

Acrosome↗
PubMed1989

Methods for evaluating the acrosomal status of mammalian sperm.

A full understanding of the acrosome reaction is central to understanding sperm function. Acrosomal status can be determined on living, motile sperm in only a few mammalian species. For other species, many light microscopic methods have been developed, including colored stains for bright-field microscopy, and probes for fluorescence microscopy. We review the existing methods and the criteria that should be considered in the choice of an assay.

Acrosome↗
PubMed1989

The physiology of sperm recovered from the human cervix: acrosomal status and response to inducers of the acrosome reaction.

Cervical mucus was collected from 35 women after artificial insemination. Mucus collections were performed at 1 h, 1 day, 2 days, or 3 days following insemination. Sperm viability was greater than 80% at all recovery times as assessed by exclusion of the supravital dye Hoechst 33258. Virtually 100% of the viable sperm were acrosome-intact at all times as assessed with a fluorescein isothiocyanate-conjugated pea lectin. Sperm were recovered from the mucus after migration into the Biggers, Whittin, and Whittingham medium in vitro. Sperm did not undergo the acrosome reaction in response to human follicular fluid immediately after migration from the mucus but did respond to this agonist after 6 h of incubation in vitro. Sperm recovered at all times after insemination had the same pattern of response to follicular fluid. Sperm that penetrated a column of cervical mucus in vitro also responded to follicular fluid with an increase in acrosome reactions after migration from the mucus and incubation for 6 h in vitro. Unlike the sperm that migrated from cervical mucus, sperm that were separated from semen by Percoll density centrifugation did not undergo the acrosome reaction when challenged with follicular fluid after 6 h but did respond after 24 h incubation. Sperm that migrated from cervical mucus had a similar increase in acrosome reactions after 6 h incubation, regardless of whether the acrosome reaction agonist was follicular fluid or disaggregated human zona pellucida.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrosome↗
PubMed1989

A new procedure for determining acrosomal status of very small numbers of human sperm.

The acrosome of human sperm cannot be easily distinguished by light microscopy. Although several techniques are now available to label the acrosomal region of human sperm and report acrosomal status, they generally require large numbers of sperm. We describe here a new procedure in which sperm are collected and treated on small-pore filters. The acrosomal region is then labeled using fluoresceinated lectin. The main advantage of this method is that it enables the study of the acrosomal status of sperm in samples with very low sperm concentration.

Acrosome↗
PubMed1989

Correlation of acrosomal status and sperm performance in the sperm penetration assay.

The sperm of some infertile men are unable to penetrate zona pellucida-free hamster oocytes but gain that ability after treatment with human follicular fluid (hFF). We asked whether altered incidences of acrosome reacted sperm explained these observations. Patient sperm failing to penetrate oocytes had fewer acrosome reactions than did healthy males, but the percentage reacted was not correlated with oocyte penetration. Sperm incubated 3 hours, then exposed to hFF, exhibited increased penetrations for 7 of 10 males, without an increase in percentage reacted sperm. Sperm incubated 22 hours before hFF treatment had penetrating ability enhanced 250- to 1000-fold, but the percentage reacted increased only sixfold. We conclude that factors other than the percentage reacted sperm are the major determinants of penetration capacity.

Acrosome↗
PubMed1988

The repeated dose toxicity of a zinc oxide/hexachloroethane smoke.

Mice, rats and guinea pigs were exposed to the smoke produced by ignition of a zinc oxide/hexachloroethane pyrotechnic composition, 1 h/day, 5 days/week, at three different dose levels, together with controls. The animals received 100 exposures except for the high dose guinea pigs, which underwent 15 exposures, because of high death rate during the first few days of exposure. The test material had very little effect on weight gain, but there was a high rate of early deaths in the top dose of mice. A variety of incidental findings was seen in both decedents and survivors, but organ specific toxicity was, with one exception, confined to the respiratory tract. The most important of these findings was a statistically significant increase in the frequency of alveologenic carcinoma in the high dose group mice (p less than 0.01) and a statistically significant trend in the prevalence of the same tumour over all dose groups and the controls. A variety of inflammatory changes was seen in the lungs of all species and some appeared to be treatment-related. Fatty change in the mouse liver was more common in the middle and high dose groups than the controls. The aetiology of the tumour incidence is discussed and it is pointed out that hexachloroethane and zinc, as well as carbon tetrachloride, which may be present in the smoke, may be animal carcinogens in appropriate circumstances. Carbon tetrachloride is a known human carcinogen.

Animals↗