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Lynn R. Fraser

Summary. 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 ≥90 μm-Ca2. Considerably higher concentrations were required to initiate optimal sperm responses: acrosome reaction, 900 μm; gamete fusion, 900 μm; hyperactivated motility, 1·80 mm; zona penetration, 1·80 mm. None of these changes was effected when Ca2+ was <450 μm. 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+ (× 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+ (× 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.

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Lynn R. Fraser

Summary. Earlier studies have provided indirect evidence that the availability of endogenous adenosine can modulate the fertilizing ability of mouse spermatozoa during capacitation. More direct evidence has been sought by evaluating the effect of exogenous adenosine present during the early stages of capacitation. A concentration-dependent stimulation of in-vitro fertilizing ability was observed, with 10 μm- and 100 μm-adenosine significantly increasing the proportion of eggs fertilized compared with drug-free controls. The adenosine-induced stimulation was observed in the presence of 0·01 μm- and 0·1 μm-dipyridamole, an inhibitor of adenosine uptake, suggesting that adenosine is acting at an external site. Comparison of adenosine with its analogues 2′-deoxyadenosine and 2-chloroadenosine indicated that the analogues at 10 μm were able to stimulate fertilization in a manner similar to adenosine. While neither adenosine nor 2′-deoxyadenosine was consistently effective at 1 μm, 2-chloroadenosine significantly stimulated fertilization at both 1 μm and 0·1 μm. In addition, 5′-N-ethylcarboxamidoadenosine (NECA) and (R)-N6-phenylisopropyladenosine (R-PIA), potent analogues in somatic cell systems, proved to be so with mouse sperm suspensions, NECA being stimulatory at ≥0·01 μm and R-PIA at ≥0·1 μm. Subjective evaluation of motility patterns indicated that more cells exhibited hyperactivated motility in the presence of stimulatory concentrations of adenosine or analogues. Assessment of capacitation state using chlortetracycline fluorescence patterns indicated that incubation in 2′-deoxyadenosine resulted in significantly fewer cells expressing the uncapacitated F pattern and significantly more cells with the capacitated AR (acrosome-reacted) pattern, compared with drug-free counterparts. It is concluded that adenosine promotes capacitation by interacting with externally-directed receptors, possibly on adenylate cyclase to increase the intracellular availability of cyclic adenosine monophosphate (cAMP); cAMP is known to stimulate mouse sperm fertilizing ability. The greater sensitivity to NECA, 2-chloroadenosine and R-PIA, relative to adenosine and 2′-deoxyadenosine, is consistent with interaction at stimulatory A2 adenosine receptors.

Keywords: adenosine; adenosine analogues; capacitation; A2 adenosine receptors; NECA; R-PIA;

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Lynn R. Fraser

Summary. 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.

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Lynn R. Fraser

Summary. Evidence is presented to indicate that older mouse eggs are fertilized more rapidly and also develop more rapidly to the pronuclear stage than younger ones. After a low (1·5 i.u.) or a high (7·5 i.u.) dose of PMSG, female mice were killed 13 or 17 h after hCG. The eggs were mixed in vitro with preincubated spermatozoa and fixed 1–1¼ h later. Although fertilization levels were high in all groups, the stages of egg activation and sperm head decondensation differed significantly. The observed ranking, from most to least rapid fertilization, of eggs obtained from females treated with 7·5 i.u. and killed 17 h after hCG, 1·5 i.u. and 13 h, and 7·5 i.u. and 13 h, was consistent with the approximate length of time the eggs had resided in the oviduct, i.e. the longer that time period, the more rapid the fertilization. When eggs were fixed 4¼ h after mixing with spermatozoa, the majority of older eggs were fully pronuclear while only a few of the younger eggs were as advanced, indicating accelerated nuclear development in the cytoplasm of the older eggs.

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Lynn R. Fraser

Summary. Concentrated suspensions of epididymal spermatozoa obtained from two strains of mice, TO and C57BL/10, were preincubated for 20 min, 1 h or 2 h before dilution and addition of (C57BL/10 × CBA)F1 eggs. While all 3 groups of TO spermatozoa demonstrated high fertility (>90% of eggs subsequently cleaved), C57BL/10 spermatozoa preincubated for 20 min and 1 h gave significantly reduced fertilization rates compared with those in the 2 h group. Furthermore, the penetration rate of the C57BL/10 spermatozoa preincubated for 20 min was significantly slower than that for similarly treated TO spermatozoa, as demonstrated by a delay in the first cleavage. A long preincubation of C57BL/10 spermatozoa in a diluted rather than a concentrated suspension did not improve fertility, suggesting that optimal capacitation may be sperm concentration-dependent in some, if not all, strains of mice.

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Lynn R. Fraser

Summary. Albumin was required specifically for penetration of the zona pellucida (<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.

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Lynn R. Fraser

Summary. Evidence for accelerated mouse sperm penetration into eggs in vitro, and hence for accelerated capacitation, was obtained when the fertilizing ability of sperm suspensions was tested after preincubation, for a period of time insufficient to permit full capacitation under standard conditions, in the presence or absence of caffeine. When eggs were fixed after 1 h, the majority (76·7%) in the caffeine-containing medium were fertilized compared with only 32·1% in the caffeine-free medium. When fixation was later, after 1 h 15 min, fertilization levels were high (approximately 90%) in both groups, but significant differences in the stages of egg activation and sperm head decondensation reached were observed. In the absence of caffeine, early stages of nuclear development were predominant, while in the presence of caffeine, a significant proportion of eggs had reached terminal stages of activation. There was also some evidence for precocious sperm, but not egg, nuclear development when fertilization occurred in caffeine-containing medium.

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Lynn R. Fraser

Summary. 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.

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Lynn R. Fraser

Summary. 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 (106 cells/ml) showed high rates of fertilization with zona-free eggs (100–95·3%); with the lower concentration of 105 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.

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Lynn R. Fraser

Summary. 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 105 and 106/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.