In vitro capacitation and fertilization.
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Animals↗
Biomedical subjects
L R Fraser
Publications and source records attributed to L R Fraser.
Cyclic AMP-dependent phosphorylation of epididymal mouse sperm proteins during capacitation in vitro: identification of an M(r) 95,000 phosphotyrosine-containing protein.
Cyclic AMP-dependent changes in phosphorylation of epididymal mouse sperm suspensions were examined in media designed to manipulate capacitation and the expression of parameters associated with full fertilizing ability, i.e. hyperactivated motility and the acrosome reaction. After initial assessment of cAMP-dependent protein kinase activity in frozen-thawed and lyophilized sperm suspensions using exogenous substrate, phosphorylation of endogenous sperm phosphoproteins was examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by autoradiography or immunoblotting. Numerous phosphoproteins were detected in both incapacitated and capacitated suspensions, the majority of which were probably concerned with motility; full expression of fertilizing ability appeared to involve an increase in the amount of endogenous phosphorylation as deduced from the decreased amount of 32P incorporation in these suspensions. The addition of the cAMP-dependent protein kinase inhibitors, H8 and PKI (6-22) amide, demonstrated that most of the phosphoproteins detected were phosphorylated in a cAMP-dependent manner. Of particular interest was a phosphoprotein with an M(r) of about 95,000 which was consistently observed in capacitated suspensions. Evidence suggests that this may be phosphorylated on tyrosine residues, since the inclusion of orthovanadate, a phosphoryltyrosine phosphatase inhibitor, altered phosphorylation of this protein. Furthermore, immunodetection using the antiphosphotyrosine antibody, PY-20, identified five proteins with approximate M(r) 116,000, 105,000, 95,000, 86,000, and 76,000, and possibly a sixth at 54,000. The 95,000 protein was consistently diminished in ionophore-treated spermatozoa, indicating that the protein was located in the acrosomal cap region. These results suggest that the protein may be the same phosphotyrosine-containing protein as that described by Leyton and Saling (1989) which has been proposed to play a role in acrosomal exocytosis.
Acrosome↗
Na(+)-requiring mechanisms modulate capacitation and acrosomal exocytosis in mouse spermatozoa.
Mouse spermatozoa require extracellular Na+ for both capacitation and acrosomal exocytosis, but the minimum concentrations differ widely: > 1 < or = 25 mmol Na+ l-1 will support capacitation, but > 125 mmol Na+ l-1 is needed for acrosomal exocytosis in capacitated cells. Our conclusions are based on evidence obtained from sperm cells preincubated in iso-osmotic media with differing concentrations of Na+ and then analysed for occurrence of the acrosome reaction, capacitation-related changes in chlortetracycline (CTC) fluorescence and in vitro fertilization. The modified Tyrode's medium used as the control medium in these experiments contained 150 mmol Na+ l-1 and supported full sperm function. At least some of the Na+ needs to be internalized to promote the functional changes, as evidenced by the ability of the monovalent cation ionophore monensin to accelerate capacitation and trigger acrosomal exocytosis in control medium. However, in low Na+ (25 mmol l-1) medium, monensin could only modulate the transition to the capacitated state, assessed with CTC, indicating that higher concentrations of extracellular Na+ are required for initiation of acrosomal exocytosis. We suggest that changes in the composition of the female reproductive tract fluids serve to control expression of sperm functional potential. Before ovulation in the mouse, sufficient Na+ and Ca2+ are present to promote capacitation. However, the Na+ concentration is marginal for support of acrosomal exocytosis and the relatively high K+ reinforces an inhibition of exocytosis. At ovulation, the release of follicular fluid would increase the Na+ and decrease the K+ concentrations, thereby permitting full expression of fertilizing potential. Possible mechanisms that might be involved in the Na(+)-related responses, including a Na(+)-Ca2+ exchanger, a Na(+)-K+ ATPase and a Na(+)-H+ exchanger, were also investigated. If a Na(+)-Ca2+ exchanger has a role to play, it is not during capacitation per se. Incubation of sperm cells in high Na+, low Ca2+ (90 mumol CaCl2 l-1) medium that supports capacitation, followed by introduction of monensin, which would have promoted an influx of Na+ and could have, in turn, activated a Na+ out, Ca2+ in response, did not accelerate transition to the capacitated state (B pattern of CTC fluorescence). In contrast, it is possible that a Na(+)-K+ ATPase may play a role during capacitation. Incubation of suspensions in control medium plus ouabain, which would inhibit the ATPase, significantly accelerated the transition from the incapacitated to the capacitated state, although it did not trigger acrosomal exocytosis.(ABSTRACT TRUNCATED AT 400 WORDS)
Acrosome↗
Adenosine analogues with specificity for A2 receptors bind to mouse spermatozoa and stimulate adenylate cyclase activity in uncapacitated suspensions.
Adenosine and its analogues, known to stimulate adenylate cyclase activity in somatic cells via A2 receptors, can accelerate capacitation in mouse spermatozoa and thereby enhance fertilizing ability in vitro. Indirect evidence has suggested that adenosine can modulate mouse sperm adenylate cyclase, implicating this enzyme and cAMP in the observed functional responses. In the present study we provide evidence that [3H]5'-N-ethylcarboxamidoadenosine (NECA), an adenosine analogue with specificity for stimulatory A2 adenosine receptors, can bind to mouse spermatozoa. This binding can be displaced by both unlabelled NECA and 2-chloroadenosine, another A2 receptor agonist, but not by cyclopentyladenosine, an inhibitory A1 receptor agonist, suggesting that the NECA binding is specific for A2 receptors. The presence of S-(p-nitrobenzyl)-6-thioinosine, an adenosine transport inhibitor, did not affect binding, indicating an external site for interaction with sperm cells. Saturable specific binding of [3H]NECA to mouse spermatozoa incubated at 37 degrees C was observed, with a Bmax of 5.17 pmol mg-1 protein and a Kd value of 930 nmol l-1. Binding data were consistent with the presence of a single major class of receptor. In addition to demonstrable binding of [3H]NECA, both NECA and 2-chloroadenosine significantly stimulated adenylate cyclase activity in a concentration-dependent manner, with NECA being effective at a lower concentration. Furthermore, the hydrolysis-resistant GTP analogue Gpp(NH)p, alone and in the presence of either NECA or 2-chloroadenosine, also significantly stimulated enzyme activity. In somatic cells, expression of responses to adenosine usually requires GTP and G proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
2-Chloroadenosine↗
Ca(2+)-related changes in the capacitation state of human spermatozoa assessed by a chlortetracycline fluorescence assay.
Chlortetracycline (CTC) fluorescence patterns were used to assess Ca(2+)-related changes in the capacitation state of human spermatozoa incubated under conditions that would affect their intracellular Ca2+ levels. Initial experiments were designed to identify consistently occurring patterns and to correlate these with acrosomal status. Incubation for up to 1 h with the ionophore A23187 (10 mumol l-1), known to promote capacitation and acrosomal exocytosis, allowed the identification of three different CTC staining patterns which were very similar to those described for mouse spermatozoa. For this reason, they were given the same nomenclature: 'F'-characteristic of uncapacitated, acrosome-intact cells; 'B'-characteristic of capacitated, acrosome-intact cells; and 'AR'-characteristic of capacitated, acrosome-reacted cells. The distribution of the three patterns in the ionophore-treated suspensions was very different from that in control suspensions treated with dimethylsulfoxide only, with a significantly higher proportion of cells displaying the B and AR patterns and a significantly lower number of cells displaying the F pattern in the ionophore-treated group at all times. A strong concordance was found between the acrosomal status of cells determined using both CTC and fluorescein-conjugated Pisum sativum agglutinin (PSA) staining methods on the same cells. Verification of PSA staining patterns with acrosomal status was obtained by means of transmission electron microscopy. The proportion of cells with uniform fluorescence in the acrosomal region correlated with acrosome-intact cells; those with only equatorial segment staining correlated with fully-reacted cells, and those exhibiting equatorial fluorescence and patchy fluorescence over the rest of the acrosomal region correlated with cells in intermediate stages of exocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)
Calcimycin↗
A biphasic pattern of 45Ca2+ uptake by mouse spermatozoa in vitro correlates with changing functional potential.
Mouse sperm capacitation in vitro, leading to hyperactivated motility, acrosomal exocytosis and rapid fertilization, takes approximately 120 min in a medium containing sufficient Ca2+. During that period, spermatozoa incubated in 45Ca2+ exhibited a biphasic pattern of Ca2+ uptake, with the first and lower peak occurring from 10 to 50 min and the second and higher peak from 60 to 90 min. When the exogenously supplied glucose was reduced from 5.56 mmol l-1 to 5.56 mumol l-1, the latter supporting capacitation but not fertilization, only the first peak of 45Ca2+ uptake was observed. Increasing the glucose to a millimolar concentration produced a second peak of uptake. We therefore propose that the first phase of 45Ca2+ uptake is associated with capacitation and the second phase with acrosomal exocytosis, which are both necessary prerequisites for fertilization. In micromolar glucose the rate of 45Ca2+ uptake during the first 30 min was 47% higher than in millimolar glucose, suggesting that the former conditions might promote a precocious rise in the intracellular Ca2+ concentration ([Ca2+]i) and hence accelerate capacitation. This hypothesis was confirmed by demonstrating both significantly accelerated transition from the uncapacitated F pattern of chlortetracycline (CTC) fluorescence to the capacitated B and AR patterns and significantly higher fertility in vitro in suspensions preincubated for 30 min in micromolar glucose, compared with those maintained continuously in millimolar glucose. These results suggest that an ATP-dependent mechanism, for example a Ca(2+)-ATPase, may be involved in maintaining a low [Ca2+]i. In micromolar glucose, available ATP would be limited and hence the ATPase activity would decline, allowing [Ca2+]i to rise.(ABSTRACT TRUNCATED AT 250 WORDS)
Acrosome↗
Ca(2+)-related changes in the mouse sperm capacitation state: a possible role for Ca(2+)-ATPase.
Mammalian spermatozoa require extracellular Ca2+, some of which must be internalized, to undergo complete capacitation. At a critical threshold, a rise in intracellular Ca2+ will trigger acrosomal exocytosis. We used chlortetracycline (CTC) fluorescence patterns to assess changes in the capacitation state of mouse spermatozoa after incubation under various conditions that would affect their intracellular Ca2+ concentrations. Under standard conditions with 1.80 mmol CaCl2l-1 known to support capacitation within 120 min and subsequent fertilization in vitro, a rise in the number of capacitated, acrosome-intact cells (B pattern) was observed over the first 60 min, followed by a decline. A detectable increase in capacitated, acrosome-reacted cells (AR pattern) coincided with the maximum of B pattern cells and a continued rise was observed over the following 60 min. With incubation in 3.60 mmol Ca2+l-1, the rise in AR cells began at 30 min, suggesting that this treatment accelerates capacitation. Introduction of ionophore A23187 at 15 min to cells in standard Ca2+ produced a similar but even more rapid response, with a maximum in B pattern cells and a noticeable rise in AR cells within 10 min. Thus ionophore-treated cells proceed through capacitation, but do so very quickly. However, ionophore in the presence of 90 mumol Ca2+l-1 could promote transition from the uncapacitated F pattern to the capacitated B pattern, but could not trigger acrosomal exocytosis, indicating that the latter requires high extracellular Ca2+. After preincubation in Ca(2+)-deficient medium, most cells exhibited the uncapacitated F pattern and the introduction of millimolar Ca2+ altered this distribution only slowly, over a period of 50 min. In contrast, preincubation in 90 mumol Ca2+l-1 resulted in a minority of F pattern cells and, within 10 min of millimolar Ca2+ introduction, a significant increase in AR cells was observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Requirements for successful mammalian sperm capacitation and fertilization.
The function of sperm is to fertilize oocytes, thereby providing necessary genetic information and triggering oocyte activation and subsequent metabolic activities that are vitally necessary for successful mammalian embryonic development. Although motile when they leave the male reproductive tract, mammalian sperm are nonfertilizing; they require a further period to complete the maturation initiated in the epididymis. This final maturation phase is called capacitation and is absolutely crucial for successful sperm:oocyte interaction. Capacitation, which exhibits a species-dependent time requirement, involves loss of proteins from the sperm surface; additionally, it has been shown to be dependent on the composition of the environment. Adequate free calcium (Ca2+), sodium (Na+), and potassium (K+), and energy substrates play roles in regulating capacitation, hyperactivated motility, the acrosome reaction, and fusion with the oocyte plasma membrane. In general, conditions present within the female reproductive tract are permissive, ie, appropriate levels of the constituents are present. Therefore, the failure to achieve conception in some couples suggests that there may well be basic physiological defects in the sperm of the male partner. Current research in many laboratories is directed toward identifying such individuals and, in time, developing appropriate therapeutic treatments.
Animals↗
Expression of capacitation-dependent changes in chlortetracycline fluorescence patterns in mouse spermatozoa requires a suitable glycolysable substrate.
Chlortetracycline (CTC) fluorescence patterns were assessed in epididymal mouse sperm suspensions capacitated in exogenous substrate-containing and substrate-free media. A capacitation-dependent transition from a majority of acrosome-intact cells expressing the uncapacitated F pattern of fluorescence to a majority with the capacitated acrosome-intact B and acrosome-reacted AR patterns was confirmed for suspensions incubated a total of 120 min in the presence of a glycolysable substrate, glucose. In contrast, assessment of spermatozoa incubated for 120 min in substrate-free medium revealed a majority of cells with the uncapacitated F pattern, despite an earlier demonstration that such cells are essentially capacitated: upon the introduction of glucose, suspensions are immediately highly fertile. When a suitable glycolysable substrate, either glucose or mannose but not fructose, was added to such suspensions, the distribution of CTC patterns changed within 10 min to a majority of B and AR patterns. Furthermore, the degree of change from uncapacitated to capacitated patterns was substrate concentration-dependent. In contrast, the introduction of the non-metabolizable substrates 2-deoxyglucose and 3-0-methylglucose and the oxidizable substrates sodium pyruvate and sodium lactate caused no change in the patterns from those seen in substrate-free medium. The in-vitro fertilizing ability of sperm suspensions to which increasing amounts of glucose or mannose were added, after initial substrate-free preincubation, directly paralleled the changes in CTC patterns and was as rapid as for suspensions incubated continuously in either hexose. We therefore conclude that the alteration in position of surface components to which CTC binds is not only capacitation-dependent, but also energy-dependent. In the absence of an appropriate exogenous glycolysable substrate, the final transition cannot occur, even though the cells are essentially capacitated.
Acrosome↗
Characterization of a decapacitation factor associated with epididymal mouse spermatozoa.
Earlier studies demonstrated that epididymal mouse spermatozoa have a surface-associated factor which inhibits fertilizing ability in a reversible manner. The factor can be removed from uncapacitated spermatozoa by gentle centrifugation, resulting in immediately highly fertile gametes, and it can be added back to capacitated spermatozoa, resulting in poorly fertile cells in which the acrosome reaction has been blocked. Using such inhibition of in-vitro fertilizing ability as an assay, we have carried out experiments to characterize the factor. It appears to be an anionic polypeptide with Mr of approximately 40,000 (according to its behaviour on gel filtration). It is stable to heating at 100 degrees C for 15 min and is not destroyed by proteases at pH 8.0, yet inhibitory activity decreases during sperm incubation in capacitating conditions and is also destroyed in partially purified preparations by endogenous enzyme action during incubation at pH 5.0. Activity is not adsorbed to either concanavalin A-agarose or wheat-germ agglutinin-agarose, suggesting that terminal mannose and N-acetylglucosamine residues are not abundant. The factor causes rapid changes in the patterns of chlortetracycline fluorescence seen on sperm heads, a parameter used to assess the capacitated state. Removal of the factor from uncapacitated cells results in a shift to a predominance of capacitated patterns, while the addition of crude or partially purified factor to capacitated cells inhibits the acrosome reaction and causes a shift to the uncapacitated pattern in acrosome-intact spermatozoa. The factor therefore behaves as a decapacitation factor. However, it appears to differ from other characterized decapacitation factors in terms both of molecular size and of abundance of mannose and N-acetylglucosamine residues.
Acrosome↗
Adenosine and its analogues, possibly acting at A2 receptors, stimulate mouse sperm fertilizing ability during early stages of capacitation.
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 microM- and 100 microM-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 microM- and 0.1 microM-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 microM were able to stimulate fertilization in a manner similar to adenosine. While neither adenosine nor 2'-deoxyadenosine was consistently effective at 1 microM, 2-chloroadenosine significantly stimulated fertilization at both 1 microM and 0.1 microM. 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 greater than or equal to 0.01 microM and R-PIA at greater than or equal to 0.1 microM. 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.
Adenosine↗
Cyclic nucleotides and mammalian sperm capacitation.
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3',5'-Cyclic-AMP Phosphodiesterases↗
Calcium channel antagonists modulate the acrosome reaction but not capacitation in mouse spermatozoa.
Mouse spermatozoa require micromolar concentrations of calcium for capacitation but millimolar levels to initiate an acrosome reaction. Sperm suspensions were capacitated by incubation for 120 min in modified Tyrode's medium containing 90 microM-CaCl2 and then verapamil (0.5-50 microM) or nifedipine (0.1-100 nM), drugs shown to inhibit voltage-sensitive calcium channels in other cell types, was added before the introduction of 1.80 mM-CaCl2. Verapamil at 5-50 microM and nifedipine at 1-100 nM significantly inhibited the calcium-stimulated acrosome reaction response, relative to the drug-free control samples. The possibility that these compounds might inhibit calcium entry during capacitation was examined by incubating suspensions for 120 min in medium containing 90 microM-CaCl2 plus either 5 microM-verapamil or 1 nM-nifedipine, diluting to reduce drug concentration to one-tenth and then adding 1.80 mM-CaCl2. The considerable acrosome reaction response obtained indicated that spermatozoa had undergone capacitation and were able to respond to calcium, despite the continuous presence of calcium channel antagonist at a concentration able to inhibit the response at the end of capacitation. In-vitro fertilization studies indicated that both drugs significantly decreased ability of the spermatozoa to fertilize eggs, consistent with acrosome reaction data. However, results indicated that 50 microM-verapamil was able to induce initial stages of egg activation and thus prevent sperm-egg fusion in zona-intact eggs. The addition of verapamil or nifedipine to suspensions capacitated for 120 min in 1.80 mM-CaCl2 significantly potentiated the acrosome reaction response, compared with drug-free controls. Similar treatment of suspensions incubated for only 30 min, and hence only partly capacitated, failed to evoke a response. The potentiation of the acrosome reaction response by verapamil in cells capacitated in high calcium may indicate either retention, due to the action of antagonists, of released intracellular calcium stores, resulting in intracellular calcium concentrations above the threshold required to trigger the acrosome reaction or action of an activated guanine nucleotide binding (G) protein to produce an agonistic rather than an antagonistic response. These results suggest that calcium channels similar to those termed voltage-sensitive in other cell types may exist and play an important role in calcium movements at the end of capacitation, but not during earlier phases of capacitation.
Acrosome↗
Extended exposure to follicular fluid is required for significant stimulation of the acrosome reaction in human spermatozoa.
The effect of human follicular fluid (FF) on the incidence of spontaneous acrosome reactions (AR) in human spermatozoa was examined over a 24-25 h period using electron microscopy. Suspensions of motile spermatozoa were prepared by a swim-up method in Earle's medium, known to support in-vitro fertilization. After adjusting the concentration to 10 x 10(6) cells/ml, suspensions were diluted 1:1 with medium (control) or FF, the latter giving a final concentration of 50% FF. In addition, at 5 h and 24 h an aliquant of the control suspension was removed, diluted 1:1 with FF and incubated for 1 h; the three suspensions were examined at 6 h and 25 h. Continuous exposure to 50% FF stimulated the AR, the effect being significant (P less than 0.001) at 25 h. However, the 1-h short exposure of spermatozoa to FF did not produce an increase in AR, even after 24 h preincubation. In a separate series of experiments, the effect of continuous incubation for 24 h in increasing concentrations of FF was investigated. A significant linear dose-dependent effect on the AR was observed with all concentrations assessed (P less than 0.01 for 12.5% FF and P less than 0.001 for 25, 50, 75 and 100% FF, compared with FF-free control). Therefore, human FF can stimulate the AR, but only after a continuous exposure to FF. A short exposure to FF, even after 24 h preincubation, does not trigger an increased AR response.
Acrosome↗
Human oocyte-cumulus complexes stimulate the human acrosome reaction.
Oocyte-cumulus complexes were obtained, after induced ovulation, from infertile patients participating in an in-vitro fertilization programme. About 6 h after retrieval and depending on the expansion of the cumulus, 100,000 motile spermatozoa, prepared by a migration-centrifugation method, were added. After 14-18 h incubation at 37 degrees C, oocytes were examined for signs of fertilization (pronuclei and polar body formation) and then removed; spermatozoa remaining in the incubation medium were fixed for transmission electron microscopy. To provide an adequate number of cells for observation, spermatozoa from a minimum of 3-5 oocytes from the same patient were pooled. When sufficient spermatozoa were available after insemination, the remainder of the suspension was incubated at 37 degrees C and fixed along with the corresponding oocyte-incubated sample. In all, 32 sperm samples were assessed and fertilized oocytes were obtained with 29 of these. In the 24 samples in which greater than 100 spermatozoa (mean of 192) could be assessed, 32% of spermatozoa had initiated or completed the acrosome reaction. In the 15 of these 24 samples for which oocyte-free controls were available, 31% of cells were reacting or reacted, compared with 15% of cells (P less than 0.001) in the controls. In the remaining 8 samples, incubated with oocyte-cumulus complexes, less than 100 but greater than or equal to 20 spermatozoa (mean of 42) were assessed and again 32% of spermatozoa were reacted.(ABSTRACT TRUNCATED AT 250 WORDS)
Female↗
Divalent cations, capacitation and the acrosome reaction in human spermatozoa.
The extracellular Ca2+ requirements for support of capacitation and the spontaneous acrosome reaction (AR) in human spermatozoa have been evaluated. Motile suspensions were prepared using a swim-up method, incubated for up to 24 h in media of various Ca2+ concentrations, fixed and assessed for occurrence of the AR using transmission electron microscopy. Results indicated that the AR response was significantly lower after incubation in Ca2+-deficient medium (generally less than 10% reacting cells) than in 1.80 mm-Ca2+-containing medium (approximately 15%). In the latter the majority of cells were fully reacted, while in Ca2+-deficient conditions the majority were at intermediate stages of the AR. Subsequent experiments indicated that a maximum AR response required the continuous presence of millimolar Ca2+; preincubation in the presence of micromolar Ca2+ did not prepare the spermatozoa to undergo rapid AR upon increase of Ca2+ to millimolar concentrations, suggesting that capacitation requires relatively high concentrations of extracellular Ca2+. Incubation in elevated Ca2+ (3.60 mM) promoted an even greater response (mean of 24-35% reacting cells compared with 12% for 1.80 mM-Ca2+). The ability of the divalent cations Ba2+, Mg2+ and Sr2+ (each at 1.80 mM) to substitute for 1.80 mM-Ca2+ in promoting the AR was also assessed. Of these, only Sr2+ provided a response greater than that observed in unsupplemented Ca2+-deficient medium. In Sr2+ the proportion of responding cells after 24 h (approximately 13%) was similar to that obtained in Ca2+ (approximately 15%), although a majority of those in Sr2+ were at intermediate stages. In 3.60 mM-Sr2+ the response was significantly higher than that observed in both 1.80 mM-Ca2+ and 1.80 mM-Sr2+, but significantly lower than that in 3.60mM-Ca2+. Under all conditions motility was maintained at greater than 90% for 24 h. The introduction of the Ca2+ ionophore ionomycin, in the presence of 1.80 mM-Ca2+, induced the AR in a concentration-dependent but preincubation time-independent manner, with the maximum response of approximately 60% being obtained with 30 microM-ionomycin. Finally, incubation in the presence of 1.80 mM-Ca2+ and verapamil, generally considered to be a calcium channel antagonist, resulted in a concentration- and incubation time-dependent increase in the AR, the maximum response in all groups being observed only after 24 h incubation. Recent evidence from other species suggests that this may represent an agonistic interaction with calcium channels.(ABSTRACT TRUNCATED AT 400 WORDS)
Acrosome↗
Metabolic and surface events in fertilization.
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Animals↗
Inhibition of adenosine-metabolizing enzymes modulates mouse sperm fertilizing ability: a changing role for endogenously generated adenosine during capacitation.
The effect of inhibiting adenosine-metabolizing enzymes on sperm fertilizing ability was studied to investigate a possible role for endogenously generated adenosine in the regulation of capacitation. The compounds used have been shown to be effective inhibitors of the relevant enzymes in similarly incubated mouse sperm suspensions. Inhibition of 5'-nucleotidase activity with alpha, beta-methylene adenosine 5'-diphosphate (AMPCP), to reduce available endogenous adenosine, caused a dose-dependent inhibition of the fertilizing ability of partially capacitated spermatozoa, which was significant with 100 and 250 microM AMPCP. Conversely, inhibition of adenosine deaminase with 100 nM coformycin, to increase available endogenous adenosine, promoted the fertilizing ability of partially capacitated spermatozoa when the fertilization rate of control suspensions was low. However, coformycin had no effect on sperm suspensions with moderate fertilizing ability, and it inhibited fertilizing ability when added to capacitated spermatozoa. These data are consistent with a promotion of the early stages of capacitation by endogenously generated adenosine and suggest that sensitivity to adenosine changes as capacitation proceeds. Because the majority of adenosine-metabolizing enzyme activity residues in or is directed toward the extracellular compartment in such suspensions, these effects of adenosine may be mediated at the outer surface of the cell. By interacting with receptors on adenylate cyclase, externally produced adenosine could modulate intracellular levels of cyclic adenosine monophosphate (cAMP), thereby influencing fertilizing ability.
5'-Nucleotidase↗