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L R Fraser

Publications and source records attributed to L R Fraser.

At least 73 records · Page 4Linked to original sources
PubMed1988

Enzymes of adenosine metabolism in mouse sperm suspensions.

The enzymes of adenosine metabolism were investigated in suspensions of epididymal mouse spermatozoa incubated under conditions which support capacitation in vitro. High levels of adenosine deaminase activity were found in sperm suspensions, but the enzyme was located in the surrounding medium and was not intrinsic to spermatozoa. 5'-Nucleotidase was also present in the surrounding medium while in sperm cells it existed as an ecto-enzyme. Adenosine was not metabolized by washed spermatozoa under conditions used for the assay of adenosine deaminase or adenosine kinase, but it was metabolized rapidly by unwashed sperm suspensions. Incubation of sperm suspensions in conditions which modulate fertilizing ability resulted in small alterations in intrinsic 5'-nucleotidase activity of spermatozoa. In contrast, the activity of adenosine deaminase was not consistently modulated by such manipulations. Adenosine deaminase and 5'-nucleotidase exhibited similar kinetic parameters to enzymes from other sources and their activities were inhibited by coformycin and alpha, beta-methylene adenosine 5'-diphosphate, respectively. These studies highlight the low adenosine-metabolizing ability of spermatozoa coupled with the extensive metabolism in the medium which surrounds them. Extracellular adenosine metabolism can therefore occur and may modulate capacitation in vitro.

5'-Nucleotidase↗
PubMed1987

Phosphodiesterase activity of mouse sperm incubated under conditions that modulate fertilizing potential in vitro.

Cyclic AMP has been implicated as a regulator of capacitation, but the control of its metabolism in sperm remains obscure. A recent study of mouse sperm has shown capacitation-related changes in the activities of both adenylate cyclase, which increased during incubation, and cyclic nucleotide phosphodiesterase, which decreased. The present study was conducted to extend these observations by measuring phosphodiesterase activity in sperm incubated in media with modified calcium and/or glucose content, conditions known to modulate fertilizing ability. Phosphodiesterase activity of sequential sperm samples, taken first when sperm are essentially uncapacitated and then when they are either partially or completely capacitated, decreased with time under all conditions, and in each case the greater fall in activity was seen in the medium that would support the greater change in fertilizing ability of the sperm population. Sperm washed by centrifugation to remove epididymal fluid also displayed a reduction in phosphodiesterase activity with time. The medium surrounding the sperm contained about half of the total phosphodiesterase activity, as well as 5'-nucleotidase and adenosine deaminase. The crude enzyme preparation showed complex kinetic behavior when assayed over a range of cAMP concentrations, but the reduction in activity with time was seen at all substrate levels. The observed changes in phosphodiesterase activity, together with the increased adenylate cyclase activity seen under these sperm incubation conditions, would increase cAMP availability with time, thus providing further evidence for a fundamental role for cAMP in controlling the events of capacitation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1987

Strontium supports capacitation and the acrosome reaction in mouse sperm and rapidly activates mouse eggs.

Extracellular Ca2+ is required for capacitation and fertilization in the mouse, but very little is known about the ability of other divalent cations to substitute for Ca2+. In this study, Sr2+, Ba2+, and Mg2+ were evaluated for their ability to support capacitation, the acrosome reaction, hyperactivated motility, and fertilization. Ba2+ proved to be ineffective, but Mg2+-containing medium was able to support capacitation to a greater extent than unsupplemented Ca2+-deficient media; despite this, Ca2+ was required for fertilization. In contrast, Sr2+ proved capable of substituting for Ca2+ in all events. Furthermore, Sr2+-induced responses were indistinguishable from the corresponding Ca2+-induced ones: Sperm capacitated at the same rate and underwent the acrosome reaction to the same extent. However, demonstration of sperm:egg fusion in Sr2+ required the use of zona-free eggs. This was due not to the inability of the sperm to penetrate the zona but to the very rapid activation and cortical granule release by eggs in response to Sr2+. When zona-intact eggs were used, the block to polyspermy had been mounted by the time sperm had penetrated the zona. A 15 min exposure to Sr2+ was sufficient to block sperm fusion, but a longer exposure was required to ensure the resumption of meiosis in eggs; such a response was surprising in that the eggs were freshly ovulated and not susceptible to activation by many different treatments. Thus Sr2+ can profoundly affect both gametes in the mouse: It substitutes completely for Ca2+ in sperm responses and rapidly activates eggs, possibly by displacing Ca2+ from intracellular stores into the cytoplasm, where the Ca2+ can then trigger the various events of activation.

Acrosome↗
PubMed1987

The acrosome reaction in human sperm from men of proven fertility.

Suspensions of motile sperm were prepared from semen samples donated by 40 men of recent, proven fertility and incubated under capacitating conditions for 24 h. At selected time-points aliquots were removed, assessed for motility, fixed and examined with the electron microscope to determine the rate of acrosome loss. Data indicate that acrosome loss increases significantly with time, but absolute values are relatively low. After 24 h a mean of 15.4% sperm had initiated the acrosome reaction; this figure included 9.7% which had completed it. The proportion of cells at intermediate stages was similar throughout incubation (approximately 5%), indicating that initiation of the acrosome reaction occurs at a fairly constant rate. In four samples motility declined over 24 h and in six, contaminating cells were observed. In the majority of these 10, acrosome loss was higher than that observed in the remaining 30 samples. Additionally, the assessment of greater than 25,000 cells during this study made it possible to evaluate specific ultrastructural features of the normal acrosome reaction in human sperm. Six stages were identified, with the intermediate ones involving loss of acrosomal matrix material while outer membranes appear to retain their integrity; this contrasts sharply with the current view of the generalized mammalian sperm acrosome reaction.

Acrosome↗
PubMed1987

Minimum and maximum extracellular Ca2+ requirements during mouse sperm capacitation and fertilization in vitro.

The minimum and maximum extracellular Ca2+ concentrations required to promote capacitation, the acrosome reaction, hyperactivated motility, zona penetration and gamete fusion in the mouse have been established. The traces of free calcium in Ca2+-deficient medium were shown not to enhance capacitation since the inclusion of EGTA to chelate free ions during a 120 min preincubation failed to alter the kinetics of capacitation from those observed in the absence of EGTA; 1 h after addition of 1.80 mM-Ca2+, both suspensions were highly fertile. Complete capacitation, when suspensions were immediately functional upon the addition of 1.80 mM-Ca2+, required the presence of greater than or equal to 90 microM-Ca2. Considerably higher concentrations were required to initiate optimal sperm responses: acrosome reaction, 900 microM; gamete fusion, 900 microM; hyperactivated motility, 1.80 mM; zona penetration, 1.80 mM. None of these changes was effected when Ca2+ was less than 450 microM. The responses to elevated Ca2+ were dependent on the length of incubation, being initially positive and then negative. A short (30 min) exposure to 3.40 mM-Ca2+ (x 2 the standard) accelerated capacitation, as evidenced by significantly increased acrosome loss, precocious expression of hyperactivated motility and enhanced fertilizing ability when Ca2+ was reduced to 1.80 mM. However, extended (120 min) preincubation irreversibly damaged sperm function. In the presence of 7.20 mM-Ca2+ (x 4), fertilizing ability was inhibited at both 30 and 120 min, despite a high incidence of acrosome loss. The primary deleterious effect appeared to be on motility which was judged to be more erratic than in 1.80 mM-Ca2+, possibly due to elevated intracellular Ca2+. Because of the considerable difference in threshold Ca2+ concentrations, it is now possible to dissociate the Ca2+-dependent events of capacitation from those of the acrosome reaction and motility changes.

Animals↗
PubMed1987

Capacitation- and fertilization-related alterations in mouse sperm oxygen consumption.

On the basis of experiments performed some years ago, it has been generally accepted that mammalian sperm metabolic activity, as determined by manometric measurement of O2 uptake, increases during capacitation. However, when mouse sperm suspensions were incubated for a total of 120 min in glucose-containing medium which promotes both capacitation and fertilization, O2 consumption, measured with an O2 electrode at 30 and 120 min, consistently declined with time. This same pattern was also observed when pyruvate was the sole exogenous energy substrate, despite the fact that these cells, although capacitated, cannot fertilize eggs until supplied with glucose. Since mouse spermatozoa require a suitable glycolysable substrate to express full functional ability, these results suggest that a shift in metabolic activity from oxidative phosphorylation to glycolysis normally accompanies the onset of fertilizing ability in this species. It was further demonstrated that this pattern, in the presence of glucose, can be markedly modified by alterations in the medium composition which have been shown to modulate fertilizing ability. All conditions chosen inhibit fertilization while retarding (Ca2+-free), accelerating (hyperosmotic high Na+) or nor affecting (iso-osmotic high K+) the rate of capacitation. When suspensions were assessed in these media, O2 consumption increased rather than decreased over the 120 min, despite the continuous presence of glucose. When these same suspensions were assessed at 30 and 120 min in media altered to permit expression of fertilizing ability, the pattern observed was one of declining O2 consumption.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Adenylate cyclase activity of mouse sperm during capacitation in vitro: effect of calcium and a GTP analogue.

Earlier studies have shown increased adenylate cyclase (AC) activity in epididymal mouse sperm incubated under capacitating conditions in vitro. The present study investigated the effect on AC activity of excluding calcium and/or glucose from the sperm incubation medium, which would modulate expression of fertilizing potential. AC activity was higher in sperm incubated for 120 than 30 min, and was higher in sperm incubated in calcium-containing than calcium-free media for all except acrosome-reacted populations. Calcium added at the time of assay stimulated AC activity, the degree of this response being independent of the functional state of the sperm population. The guanine nucleotide analogue Gpp(NH)p slightly enhanced AC activity, but did not alter the stimulatory effect of calcium. Since calcium can increase AC activity, possibly by interaction with a divalent cation allosteric site on the catalytic subunit of the enzyme, a rise in intracellular calcium levels during capacitation may mediate the increased activity of AC, allowing expression of cAMP dependent events which are a prerequisite for fertilization.

Adenylyl Cyclases↗
PubMed1986

Both taurine and albumin support mouse sperm motility and fertilizing ability in vitro but there is no obligatory requirement for taurine.

When mouse spermatozoa were washed immediately upon release from the epididymis, preincubated for up to 120 min in PVA-containing, albumin-free medium and assessed for their ability to fertilize cumulus-intact eggs in vitro, they were poorly fertile in comparison with their unwashed counterparts in the same medium. Fertilizing ability could be significantly improved by introducing taurine or albumin or by washing a second time at the end of preincubation. The most effective treatment was provided by the continuous presence of low concentrations (0.05-0.1 mg/ml) of BSA, similar to the amount of albumin detected in the supernatants removed during washing. There was no evidence that acrosome loss was inhibited by washing; rather, it was enhanced by the removal of a surface component which inhibits the acrosome reaction. The presence of taurine did not further increase this response. Motility, reduced in washed suspensions, was improved by the presence of taurine or albumin and experimental results suggest that this was a major factor in the improvement of fertilizing ability after introduction of these compounds. Although taurine, hypotaurine and albumin were all found in the sperm washings and thus would be present in unwashed, fertile samples, low concentrations of albumin were able to maintain full fertilizing ability. Therefore, unlike hamster spermatozoa, mouse spermatozoa would not appear to have an obligatory requirement for a motility stimulating factor such as taurine.

Animals↗
PubMed1985

Acrosome loss in human sperm incubated in vitro under capacitating conditions.

Human sperm were incubated under capacitating conditions and, at selected points up to 24 h of incubation, motile cells were fixed and assessed with the electron microscope for the presence or absence of the acrosome. Two methods of sample preparation were compared. In the first, semen samples were washed, incubated and filtered through glass beads to select motile cells before fixation. In the second, motile sperm were allowed to swim up into medium; this produced greater than 95% motile cells which were then incubated and fixed as required. In both series of experiments a significant increase in acrosome loss with time was observed (P = 0.00013), although only 10.5% of cells had lost the acrosome after 24 h. It is concluded that overt acrosome loss occurs less frequently in human sperm than in those of commonly used laboratory animals.

Acrosome↗
PubMed1985

Albumin is required to support the acrosome reaction but not capacitation in mouse spermatozoa in vitro.

Albumin was required specifically for penetration of the zona pellucida (less than 10% of eggs fertilized in the absence of albumin), but was not required for capacitation. A similar rate of capacitation was observed in the presence of albumin at concentrations ranging from 30 to 1 mg/ml, while a slightly slower rate was observed in the presence of 0.25 and 0.1 mg albumin/ml. In the absence of albumin, capacitation occurred at a rate which lagged behind that of the albumin-incubated counterparts by about 30 min; once capacitated, the addition of albumin promoted rapid sperm penetration. In albumin-free media (+/- the macromolecule PVA), sperm motility was frequently reduced, with fewer cells exhibiting hyperactivated motility, but improvements were observed after introduction of albumin. Acrosome loss was significantly lower in the absence of albumin, but within 5 min of its addition at concentrations ranging from 30 to 0.1 mg/ml to capacitated sperm suspensions, acrosome loss was stimulated and reached levels similar to those seen in control samples. Therefore, albumin can trigger the acrosome reaction in capacitated spermatozoa. It appears to act by assisting in the removal of a surface-associated inhibitory component, the presence of which stabilizes the sperm membranes and inhibits the acrosome reaction.

Acrosome↗
PubMed1984

Mouse sperm capacitation in vitro involves loss of a surface-associated inhibitory component.

The increasing fertility of epididymal mouse sperm suspensions as preincubation time is extended is accompanied by the inactivation or destruction of an inhibitory component. Alternatively, precocious removal of the component, achieved by centrifugation, leads to significant improvement in fertilizing ability. Suspensions were preincubated for a total of 120 min, with aliquants being removed at 5, 30 and 120 min. By gently washing samples and resuspending in fresh medium, the poor fertility of unwashed 5- and 30-min suspensions was increased such that 30-min washed samples did not differ significantly from fully capacitated, highly fertile 120-min unwashed control samples. When the supernatants obtained during washing of uncapacitated suspensions (5 and 30 min preincubation) were added to capacitated (120 min preincubation) populations, fertilization of cumulus-intact eggs was markedly and significantly inhibited, although fertilization of zona-free eggs was unaffected. In contrast, supernatants from capacitated suspensions were not inhibitory. When suspensions were preincubated in Ca2+-free media, both washing and exposure to hyperosmolal conditions improved fertilizing ability after addition of exogenous Ca2+, although not to the extent seen in control samples. Removal of the inhibitory component therefore increased the response of spermatozoa to Ca2+. The component was shown to be cell-associated and to inhibit the acrosome reaction in capacitated suspensions. Finally, the inhibition was shown to be reversible, with further incubation of inhibited suspensions restoring the original fertility.

Acrosome↗
PubMed1983

Mouse sperm capacitation assessed by kinetics and morphology of fertilization in vitro.

Epididymal mouse spermatozoa were preincubated for periods of 5-120 min and then tested for their ability to penetrate freshly ovulated eggs synchronously and rapidly. When zona-intact eggs were used, only suspensions preincubated for 120 min gave consistently high rates of fertilization, but suspensions preincubated for 30 min were functionally equivalent to those incubated for 120 min when used with zona-free eggs; the only major observable differences were a 15-min lag in sperm-egg interaction and an increased incidence of asynchrony with multiple sperm penetrations. A morphological study of sperm-egg interactions using zona-intact eggs indicated that, within 35 min of gamete mixing, egg microvilli could be detected by SEM in association with the fertilizing sperm head. Using conventional light microscopic examination of fixed and stained preparations, initial stages of sperm head decondensation could be detected in the majority of eggs after 45-60 min and the process was essentially completed, with the egg at the telophase-second polar body stage of meiosis II, after 75 min. Similar kinetics were observed with sperm concentrations of 10(5) and 10(6)/ml. The time required for penetration by capacitated sperm suspensions is therefore relatively short and the most accurate information regarding state of capacitation and rate of sperm penetration can be obtained by choosing an appropriately short interval for sperm-egg interaction before assessment.

Animals↗
PubMed1983

Potassium ions modulate expression of mouse sperm fertilizing ability, acrosome reaction and hyperactivated motility in vitro.

In K+-free medium, epididymal sperm suspensions, whether washed free of epididymally-derived K+ or not, were unable to penetrate washed cumulus masses; some penetration of zona-free eggs was obtained with unwashed sperm suspensions, while washed samples were generally non-fertilizing. Within 5 min of K+ introduction, however, spermatozoa were able to fertilize intact eggs rapidly and synchronously, indicating that K+ was not required for capacitation. Measurements of extracellular K+ concentrations in these experiments indicate that 0.1-0.15 mM-K+ is sufficient to support sperm: egg fusion, but concentrations greater than 0.15 mM are required for penetration of cumulus-intact eggs. When medium of normal osmolality (318 mosmol) but elevated K+/Na+ ratio (27.7 mM/125 mM) was compared with control medium (2.7/150), the former promoted lower rates of penetration after both 30 and 120 min preincubation (8 and 10%, respectively) than those obtained with control medium (45 and 95%). Upon reduction to the ratio in control media, however, the fertilizing potential of these suspensions was equivalent to control samples: relatively slow and asynchronous penetration after 30 min preincubation (50%) and rapid, synchronous penetration after 120 min (92%). Thus there was no evidence of a shortening of sperm capacitation time, but rather a suppression of fertilizing potential in the presence of elevated K+. Uterine sperm samples recovered shortly after mating gave similar results when tested in these media 30 and 120 min after release from the male tract. Preincubation of epididymal samples in high K+ (27.7 mM) hyperosmolal media (368 mosmol) for 30 min significantly shortened sperm capacitation as shown by rapid penetration of intact eggs (94%) after reduction in osmolality, but this appeared to be a non-specific effect; high Na+ (175 mM) hyperosmolal medium had a similar effect (98% of eggs fertilized). Acrosome loss and hyperactivated motility were significantly lower in media with very low or very high K+ concentrations but, after alteration to control medium values, increased to levels similar to those obtained with control samples. It is proposed that the relatively high K+ concentrations found in female tract fluids (approximately 20-30 mM) may serve to modulate fertilizing potential of spermatozoa in vivo.

Acrosome↗
PubMed1982

p-Aminobenzamidine, an acrosin inhibitor, inhibits mouse sperm penetration of the zona pellucida but not the acrosome reaction.

The effect of p-aminobenzamidine (pAB), an inhibitor of mouse sperm acrosin, on mouse sperm capacitation, motility, acrosome loss and fertility in vitro was examined using zona-intact and zona-free eggs. With intact eggs, concentrations of pAB ranging from 0.1 to 1.0 mM in the sperm preincubation medium effectively inhibited fertilization (13-0%, respectively), but these same suspensions (10(6) cells/ml) showed high rates of fertilization with zona-free eggs (100-95.3%); with the lower concentration of 10(5) cells/ml, fertilization rates of zona-free eggs decreased with increasing concentrations of pAB (100-55%). Washing of treated samples gave fertilization rates similar to control samples (87.1 and 84.6%, respectively), indicating that inhibition was reversible and that there had been no interference with the capacitation process. Whiplash motility was also observed in all samples, suggesting that the apparent inability to penetrate the zona might be due to an acrosomal defect. This was confirmed by electron microscopic examination of treated sperm samples. In high concentrations of pAB, many cells had undergone the acrosome reaction, i.e. membrane vesiculation, but acrosomal matrix dispersal was inhibited. These results are consistent, therefore, with a role for the acrosomal enzyme acrosin in matrix dispersal, but not the acrosome reaction itself, and in penetration of the zona pellucida.

Acrosin↗
PubMed1981

A glycolytic product is obligatory for initiation of the sperm acrosome reaction and whiplash motility required for fertilization in the mouse.

The substrate requirements for capacitation of epididymal mouse spermatozoa, initiation of the acrosome reaction and support of fertilization of mouse eggs in vitro were examined by assessing not only fertilization rates, but also the stages of egg activation and sperm head decondensation in fertilized eggs to monitor the temporal aspects of these processes. Although early events of capacitation did not require exogenous substrates, the fertilization process was effectively blocked at the terminal stages of capacitation in the absence of a glycolysable sugar, and addition of glucose was obligatory to initiate both the acrosome reaction and the whiplash motility associated with fertilizing ability. Once the spermatozoa had been primed by glucose, however, the removal of exogenous glucose did not block fertilization. The need for oxidative metabolism was excluded because successful fertilization could be achieved with glucose as the sole exogenous substrate under strictly anaerobic conditions or in the presence of 2.5 microM-oligomycin. We suggest, therefore, that epididymal mouse spermatozoa can be almost completely capacitated in the absence of metabolic processes simply by release into substrate-free medium. They require exposure to glucose to permit induction of the acrosome reaction and motility changes, necessary prerequisites for fertilization; such exposure is followed by rapid and synchronous penetration of eggs.

Acrosome↗
PubMed1981

Dibutyryl cyclic AMP decreases capacitation time in vitro in mouse spermatozoa.

Epididymal mouse sperm suspensions were preincubated for various times in medium containing glucose and/or dibutyryl cyclic AMP and then assessed for fertilizing ability in vitro, loss of the acrosome and motility changes. Capacitation time was significantly reduced by exposure to glucose and 0.1 mM-dbcAMP for 30 min as evidenced by early and synchronous fertilization of eggs, compared with glucose alone. Although this was accompanied by a precocious development of whiplash motility, the rate of acrosome loss in isolated sperm suspensions was not accelerated by the presence of exogenous cyclic nucleotide. Exposure of spermatozoa to 1 mM-dbcAMP in the presence of glucose resulted in very poor fertilization, but the effect could be prevented by withholding glucose until eggs were introduced; this may be due to free butyrate in the system since the inclusion of 1 mM-butyrate in glucose-containing medium had a similar inhibitory effect. Although cyclic nucleotide supported the acrosome reaction but not motility changes, no fertilization was obtained unless zonae were removed, when a low level of fertilization (30%) was observed. Both whiplash motility and acrosome loss are thus obligatory for sperm penetration of the zona and glycolytic metabolism supports both changes, perhaps by promoting endogenous generation of cyclic AMP to act as an intermediary in these two distinct phenomena.

Acrosome↗
PubMed1979

Analysis of aneuploidy in first-cleavage mouse embryos fertilized in vitro and in vivo.

First-cleavage mouse embryos, fertilized in vitro and in vivo, provide ideal material for chromosomal analysis. With the appropriate incubation in a mitotic inhibitor, syngamy is prevented and the sperm- and egg-derived chromosomes remain as separate clusters. Because the latter chromosomes undergo condensation sooner than those from the spermatozoon, the parental source of chromosome sets can be identified even without a marker chromosome. Thus these embryos can be analyzed both for the primary incidence and the parental source of a number of chromosomal anomalies, including aneuploidy. By using fertilization in vitro to obtain the embryos, the synchrony of fertilization and nuclear development is such that 80% or more of the chromosomal preparations are suitable for analysis, compared with about 50% for embryos fertilized in vivo.

Aneuploidy↗