Contraception
45:387-394,
1992
CALCIUM TRANSPORT AND Ca++-ATPase ACTIVITY IN SPERMATOZOAL PLASMA MEMBRANE VESICLES OF NIFEDIPINE-ADMINISTERED GUINEA PIGS R. Juneja, I. Gupta*, A. Wali, R.N. Chbavarti,
and S. Majumdar
Departments of *Obstetrics & Gynaecology and Experimental Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh- 1600 12 India ABSTRACT Spermatozoal plasma membrane vesicles isolated from distal portion of the epididymis and vas deferens were found to contain Ca*-activated ATPase and calcium transport activities. Nifedipine was administered at two different doses (1.0 and 2.5 mg/kg b.w./day) and the effect was observed for both short- (4 week) and long-term (12 week) period. The cellular ionic calcium content and Ca*-ATPase activity were observed to be enhanced in the drug-treated animals. The recovery studies carried out after 4 and 6 weeks of withdrawal of the drug treatment exhibited partial to complete restoration of observed changes. The stimulatory rather than inhibitory effect of Nifedipine, a specific calcium channel blocker, on calcium uptake may suggest that voltage-sensitive calcium channels may be lacking in guinea pig spermatozoa. The stimulatory effect of the drug is speculated to be either by inhibition of Na+-Ca++ antiporter or G-protein activated agonistic effect or probably due to altered physicochemical properties of the drug-treated sperm plasma membranes. INTRODUCTION It has long been recognized that spermatozoa maintain a low intracellular calcium concentration (nanomolar range) despite living in an environment of millimolar calcium concentration (1,2). The maintenance of such a steep concentration gradient indicates the presence of an efficient mechanism in spermatozoa1 plasma membrane to remove free calcium from the cytoplasm. In sea urchin sperm, Ca* entry seems to be mediated by Ca* - channels because the calcium channel blockers have been shown to inhibit the Ca* influx and acrosome reaction (3,4). Our understanding of Ca* regulation in guinea pig spermatozoa is still largely fragmentary. Ionic exchanges in guinea pig spermatozoa are probably not regulated by change in membrane potential (5) and voltage-gated channels do not appear to participate in Ca+* influx (6). Instead, a Na+-Ca’+ antiporter (7) and ion ATPases (8-lo), working alone or in conjunction, may be responsible for the regulation of the intracellular concentration of calcium. In order to have further understanding of the mechanism involved in regulation of intracellular calcium, we have elucidated the effect of Nifedipine, a specific calcium channel blocker, in the calcium build up and Ca*-ATPase activity present in guinea pig spermatozoa1 plasma membrane vesicles. Submitted Accepted
for publication for publication
August 1, 1991 March 9, 1992
Copyright
Q 1992 Butterworth-Heinemann
Contraception
388 MATERIALS
AND METHODS
Chemicals: MOPS, EDTA and Ouabain were purchased from Sigma Chemical Co., St. Louis, MO, USA. 45Ca-CaC1, was purchased from Amersham Co., New England. Drug treatment Adult male guinea pigs from the Institute’s Central Animal House facility with body weight = 500 g were maintained in the laboratory for one month on pellet diet (Lipton India Ltd., Bombay, India) for acclimatization. Water was given ad Zibitum and ascorbic acid was supplemented to all the animals. The normal control group had 10 animals whereas 4 groups (I-IV) of 30 animals each were divided into 3 subgroups of 10 animals each. Nifedipine was dissolved in ethanol and then further diluted in 0.9% NaCl. All containers were kept wrapped in aluminium foil to protect it from the light. Daily dose was given to animals by cannula under yellow sodium illumination. Control animals were given the same concentration of absolute ethanol and 0.9% NaCl. The drug was administered orally at 1.0 and 2.5 rngflcg b.w./day dose in 0.5 ml volume for both 4 and 12 weeks. After the completion of treatment, 7-8 animals from each experimental group, along with the controls were killed. The remaining animals were allowed to recover after withdrawal of the drug and killed at intervals of 4 and 6 weeks. Preparation of sperm plasma membmne vesicles: The membrane preparations were based on the method described by us earlier (11). The membrane preparation showed 5-7 fold enrichment in comparison to the whole cell homogenates of the plasma (13) membrane specific marker enzyme Na+ - K+ - ATPase (12) and 5’-nucleotidase (Table I), thus confirming purity and ensuring enzyme activity and integrity of the isoThe protein concentration of the membrane was lated plasma membrane vesicles. estimated by the sodium dodecyl sulphate-Lowry procedure of Lees and Paxman using crystalline bovine serum albumin as the standard (14). All operations were carried out at 4°C. Table I: Enzyme activities (nmoles/min/mg protein) in the various fractions of treated guinea pig sperm
Enzyme 5’-Nucleotidase Na+,K+-ATPase Succinate dehvdrogenase
No. of sperm infusates 24 21 21
Enzyme Activity Sperm membrane Sperm vesicles infusate 5.39 f 0.82 26.00 f 1.49 9.34 + 1.78 62.39 + 3.74 10.21 f 0.28
Enrichment 4.82 6.68
1.8 f 0.02
Values are Mean & S.E.M. for number of infusate indicated. Calcium uptake: Ca++ - uptake by spermatozoa1 plasma membrane vesicles was measured by a rapid filtration method developed by Rufo et al. (15). Plasma membrane vesicles prepared from sperm infusate was incubated at 37°C for 5 min in an incubating buffer (110 mM NaCl, 5 mM KCl, 10 mM MOPS, pH 7.4). The reaction was started by the addition of 0.2 pCi/assay of 45Ca-CaC1, (Sp. activity = 21.83 mCi/ml; Amersham Corp., England). At indicated time intervals, 100 ~1 (25 /.tg protein) of the sample was removed and processed as described earlier (11). Ca*-ATPase activity: The Ca*-ATPase activity was measured as described by us earlier for human sperm plasma membrane vesicles (16). Inorganic phosphorus liberated from the reaction was determined by the method of Chen et al. (17). Estimation of calcium content: Total cellular calcium was estimated in sperm infusate by calcium analyser which incorporates a fluorometic titration (Calcetter Model 4008 & 4009, Precision systems, MA, USA). A value of 3% of total calcium content was considered to represent ionic calcium (18) which had been confirmed at the beginning of
Contraception the study. About 6-8 sperm suspensions obtained from different animals of the same experimental group were pooled together for plasma membrane preparation in a single experiment. The results of Ca+*-ATPase and other enzymes have been based on two independent membrane preparations, analyzed in triplicate. Statistical analysis: The analysis of variance was used to compare the data (19). RESULTS
Calcium uptake by membmne vesicles: The effect of Nifedipine administration on the time course of Ca*-uptake by spermatozoa1 plasma membrane vesicles is shown in Fig. 1 and 2. The sperm plasma membrane vesicles prepared from the drug-treated animals sequestered Ca’+ at a faster rate and to a greater extent than those prepared from the control group of animals. Nifedipine showed a dose-related effect on the stimulation of calcium uptake system. The stimulatory effect of the drug was more pronounced at a dose of 2.5 mg&g b.w./day (Figs. la and 2a) than the daily dose of 1.0 mgJkg b.w./day (Figs. lb and 2b). During the recovery period, the drug-treated animals showed partial to complete restoration of stimulatory levels (Figs. 1 and 2) as indicated by the reduction in calcium content also. Cacc-ATPase activity: After short-term treatment, Nifedipine exhibited a doserelated stimulation of Cace-ATPase with no recovery even after 4 to 6 weeks of withdrawal of the drug (Table 11). However, the drug remained ineffective after 1Zweek treatment of animals with a dose of 1 mg/kg b.w./day. At a daily dose of 2.5 mg/kg b.w./day, the drug resulted in a highly significant (pcO.01) increase in enzyme activity which showed a tendency of restoration after 6 weeks of recovery period (Table II).
Fig. 1: Kinetics of Ca”+-uptake by guinea pig spermatozoa1 plasma membrane vesicles . (a) Plasma membrane vesicles prepared from animals of control, Nifedipine-treated (2.5 mg/kg b.w./day for 4 weeks) and 4-week recovery experimental groups were incubated in a medium containing 110 mM NaCl, 5 mM KCI, 10 mM MOPS, pH 7.4, at 37°C. Following a 5-min preincubation, calcium (45Ca-CaC1,, Sp. Act. 1.83 mCi/nmol, Amersham) was added at a concentration of 0.2 l.tCi/assay. At the designated time points, duplicate aliquots of vesicle suspension (25 p,g protein) were withdrawn and counted as described in the text. Data are expressed as mean + S.D. of at least three determinations on two individual preparations of plasma membrane. (b) The assay was performed as above except that Nifedipine was administered at a dose of 1.O mgJkg b.w./day for 4 weeks.
390
K E
Contraception
a 4 Wks RECOVERY . AFTER lREAn.iENT 0 NORMAL
1
Fig. 2: Effect of long-term (12-week) Nifedipine treatment on the kinetics of Ca++-uptake by guinea pig spermatozoa1 plasma membrane vesicles. The drug was administered at a dose of (a) 2.5 i&kg b.w./day and (b) 1.0 mg/kg b.w./day for 12 weeks.
CeUuhr calcium content: At 1 mgjkg b.w./day dose of Nifedipine, calcium content remained unaltered but 2.5 mg/kg b.w./day dosage of Nifedipine for 12 weeks resulted in marked enhancement of calcium content without any noticeable recovery (Table II). After short-term treatment, however, increased levels of calcium were restored within 6 weeks of withdrawal of the drug Qeatment (Table II). DISCUSSION The spermatozoa1 plasma membrane Ca++ - transport system and cellular calcium content stimulated by Nifedipine has been described in the present study. In order to study the net calcium transport across the plasma membrane, the Ca” - ATPase activity was also evaluated. A number of reports have clearly demonstrated the in vitro effect of calcium channel blockers on different spermatozoa1 characteristics (6,20). Our data with guinea pig spermatozoa demonstrates the correlation between Nifedipine administration and the net calcium transport across the sperm plasma membrane vesicles. In our assay system, Nifedipine does not exert its inhibitory action on spermatozoa1 Ca*+ build-up which it was otherwise expected to do. Several other studies have also reported that the calcium channel ligands failed to inhibit the acrosome reaction in guinea pig spermatozoa (6) and stimulated the calcium-uptake (20). Coupled with the observation that neither hyperpolarization nor depolarization initiated the acrosome reaction (5), these results suggest that the voltage-sensitive channels probably do not control calcium entry into guinea pig spermatozoa. The negligible calcium uptake observed in the control animals is in consonance with the findings of Coronel and Lardy (21). At least three explanations for the drug-enhanced calcium uptake and Ca++-ATPase activity can be put forward. Firstly, the inhibitory effect of calcium channel blockers on Na+/Ca++ antiporter activity (22) can presumably be discounted. Thus, the blocking of calcium efflux could allow the intracellular calcium concentration to rise above the threshold level.
391
Contraception Table II: Results of Nifedipine administration on different biochemical parameters in guinea pig spermatozoa Ionic calcium (nmoles/mg motein)
Ca*-ATPase (nmoles/min/ mg motein)
Control (Saline) 4-week treatment 4-week recovery 6-week recovery
3.99 f 0.22 3.83 f 0.21 3.72 f 0.75
13.82 f 1.39 15.82 + 0.75 15.82 + 0.79
Group I (I .O mglkg b.w.lday) 4-week treatment 4-week recovery 6-week recovery
4.3 1 +- 0.00 3.21 + 0.25 2.74 + 0.44**
61.03 f 1.78”” 65.99 f 1.82** 52.75 f 2.34”*
Group II 4-week 4-week 6-week
6.16 + 0.21** 4.54 + 0.24* 4.28 + 0.06
108.70 + 4.32** 87.77 f 2.74** 82.30 f 2.08**
Short-term treatment
(2.5 mglkg b.w.lday) treatment recovery recovery
Long-term treatment Control (Saline) 12-week treatment 4-week recovery g-week recovery
3.58 + 0.48 3.80 + 0.23 3.74 r 0.12
13.78 f 0.89 16.84 + 1.28 16.56 f 0.78
Group III (I .O mglkg b.w.lday) 12-week treatment 4-week recovery 6-week recovery
3.37 + 0.29 3.60 + 0.10 3.57 + 0.34
14.79 f 0.04 20.68 + 1.38 41.26 + 1.56**
Group IV (2 5 mglkg b.w ./day) 12-week treatment 4-week recovery 6-week recovery
6.89 + O.OO** 8.20 + 1.06** 6.17 f 0.14**
102.47 f 3.64** 73.25 f 2.65** 38.69 + 1.84**
“pcO.05; **p
45:387-394,
1992
CALCIUM TRANSPORT AND Ca++-ATPase ACTIVITY IN SPERMATOZOAL PLASMA MEMBRANE VESICLES OF NIFEDIPINE-ADMINISTERED GUINEA PIGS R. Juneja, I. Gupta*, A. Wali, R.N. Chbavarti,
and S. Majumdar
Departments of *Obstetrics & Gynaecology and Experimental Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh- 1600 12 India ABSTRACT Spermatozoal plasma membrane vesicles isolated from distal portion of the epididymis and vas deferens were found to contain Ca*-activated ATPase and calcium transport activities. Nifedipine was administered at two different doses (1.0 and 2.5 mg/kg b.w./day) and the effect was observed for both short- (4 week) and long-term (12 week) period. The cellular ionic calcium content and Ca*-ATPase activity were observed to be enhanced in the drug-treated animals. The recovery studies carried out after 4 and 6 weeks of withdrawal of the drug treatment exhibited partial to complete restoration of observed changes. The stimulatory rather than inhibitory effect of Nifedipine, a specific calcium channel blocker, on calcium uptake may suggest that voltage-sensitive calcium channels may be lacking in guinea pig spermatozoa. The stimulatory effect of the drug is speculated to be either by inhibition of Na+-Ca++ antiporter or G-protein activated agonistic effect or probably due to altered physicochemical properties of the drug-treated sperm plasma membranes. INTRODUCTION It has long been recognized that spermatozoa maintain a low intracellular calcium concentration (nanomolar range) despite living in an environment of millimolar calcium concentration (1,2). The maintenance of such a steep concentration gradient indicates the presence of an efficient mechanism in spermatozoa1 plasma membrane to remove free calcium from the cytoplasm. In sea urchin sperm, Ca* entry seems to be mediated by Ca* - channels because the calcium channel blockers have been shown to inhibit the Ca* influx and acrosome reaction (3,4). Our understanding of Ca* regulation in guinea pig spermatozoa is still largely fragmentary. Ionic exchanges in guinea pig spermatozoa are probably not regulated by change in membrane potential (5) and voltage-gated channels do not appear to participate in Ca+* influx (6). Instead, a Na+-Ca’+ antiporter (7) and ion ATPases (8-lo), working alone or in conjunction, may be responsible for the regulation of the intracellular concentration of calcium. In order to have further understanding of the mechanism involved in regulation of intracellular calcium, we have elucidated the effect of Nifedipine, a specific calcium channel blocker, in the calcium build up and Ca*-ATPase activity present in guinea pig spermatozoa1 plasma membrane vesicles. Submitted Accepted
for publication for publication
August 1, 1991 March 9, 1992
Copyright
Q 1992 Butterworth-Heinemann
Contraception
388 MATERIALS
AND METHODS
Chemicals: MOPS, EDTA and Ouabain were purchased from Sigma Chemical Co., St. Louis, MO, USA. 45Ca-CaC1, was purchased from Amersham Co., New England. Drug treatment Adult male guinea pigs from the Institute’s Central Animal House facility with body weight = 500 g were maintained in the laboratory for one month on pellet diet (Lipton India Ltd., Bombay, India) for acclimatization. Water was given ad Zibitum and ascorbic acid was supplemented to all the animals. The normal control group had 10 animals whereas 4 groups (I-IV) of 30 animals each were divided into 3 subgroups of 10 animals each. Nifedipine was dissolved in ethanol and then further diluted in 0.9% NaCl. All containers were kept wrapped in aluminium foil to protect it from the light. Daily dose was given to animals by cannula under yellow sodium illumination. Control animals were given the same concentration of absolute ethanol and 0.9% NaCl. The drug was administered orally at 1.0 and 2.5 rngflcg b.w./day dose in 0.5 ml volume for both 4 and 12 weeks. After the completion of treatment, 7-8 animals from each experimental group, along with the controls were killed. The remaining animals were allowed to recover after withdrawal of the drug and killed at intervals of 4 and 6 weeks. Preparation of sperm plasma membmne vesicles: The membrane preparations were based on the method described by us earlier (11). The membrane preparation showed 5-7 fold enrichment in comparison to the whole cell homogenates of the plasma (13) membrane specific marker enzyme Na+ - K+ - ATPase (12) and 5’-nucleotidase (Table I), thus confirming purity and ensuring enzyme activity and integrity of the isoThe protein concentration of the membrane was lated plasma membrane vesicles. estimated by the sodium dodecyl sulphate-Lowry procedure of Lees and Paxman using crystalline bovine serum albumin as the standard (14). All operations were carried out at 4°C. Table I: Enzyme activities (nmoles/min/mg protein) in the various fractions of treated guinea pig sperm
Enzyme 5’-Nucleotidase Na+,K+-ATPase Succinate dehvdrogenase
No. of sperm infusates 24 21 21
Enzyme Activity Sperm membrane Sperm vesicles infusate 5.39 f 0.82 26.00 f 1.49 9.34 + 1.78 62.39 + 3.74 10.21 f 0.28
Enrichment 4.82 6.68
1.8 f 0.02
Values are Mean & S.E.M. for number of infusate indicated. Calcium uptake: Ca++ - uptake by spermatozoa1 plasma membrane vesicles was measured by a rapid filtration method developed by Rufo et al. (15). Plasma membrane vesicles prepared from sperm infusate was incubated at 37°C for 5 min in an incubating buffer (110 mM NaCl, 5 mM KCl, 10 mM MOPS, pH 7.4). The reaction was started by the addition of 0.2 pCi/assay of 45Ca-CaC1, (Sp. activity = 21.83 mCi/ml; Amersham Corp., England). At indicated time intervals, 100 ~1 (25 /.tg protein) of the sample was removed and processed as described earlier (11). Ca*-ATPase activity: The Ca*-ATPase activity was measured as described by us earlier for human sperm plasma membrane vesicles (16). Inorganic phosphorus liberated from the reaction was determined by the method of Chen et al. (17). Estimation of calcium content: Total cellular calcium was estimated in sperm infusate by calcium analyser which incorporates a fluorometic titration (Calcetter Model 4008 & 4009, Precision systems, MA, USA). A value of 3% of total calcium content was considered to represent ionic calcium (18) which had been confirmed at the beginning of
Contraception the study. About 6-8 sperm suspensions obtained from different animals of the same experimental group were pooled together for plasma membrane preparation in a single experiment. The results of Ca+*-ATPase and other enzymes have been based on two independent membrane preparations, analyzed in triplicate. Statistical analysis: The analysis of variance was used to compare the data (19). RESULTS
Calcium uptake by membmne vesicles: The effect of Nifedipine administration on the time course of Ca*-uptake by spermatozoa1 plasma membrane vesicles is shown in Fig. 1 and 2. The sperm plasma membrane vesicles prepared from the drug-treated animals sequestered Ca’+ at a faster rate and to a greater extent than those prepared from the control group of animals. Nifedipine showed a dose-related effect on the stimulation of calcium uptake system. The stimulatory effect of the drug was more pronounced at a dose of 2.5 mg&g b.w./day (Figs. la and 2a) than the daily dose of 1.0 mgJkg b.w./day (Figs. lb and 2b). During the recovery period, the drug-treated animals showed partial to complete restoration of stimulatory levels (Figs. 1 and 2) as indicated by the reduction in calcium content also. Cacc-ATPase activity: After short-term treatment, Nifedipine exhibited a doserelated stimulation of Cace-ATPase with no recovery even after 4 to 6 weeks of withdrawal of the drug (Table 11). However, the drug remained ineffective after 1Zweek treatment of animals with a dose of 1 mg/kg b.w./day. At a daily dose of 2.5 mg/kg b.w./day, the drug resulted in a highly significant (pcO.01) increase in enzyme activity which showed a tendency of restoration after 6 weeks of recovery period (Table II).
Fig. 1: Kinetics of Ca”+-uptake by guinea pig spermatozoa1 plasma membrane vesicles . (a) Plasma membrane vesicles prepared from animals of control, Nifedipine-treated (2.5 mg/kg b.w./day for 4 weeks) and 4-week recovery experimental groups were incubated in a medium containing 110 mM NaCl, 5 mM KCI, 10 mM MOPS, pH 7.4, at 37°C. Following a 5-min preincubation, calcium (45Ca-CaC1,, Sp. Act. 1.83 mCi/nmol, Amersham) was added at a concentration of 0.2 l.tCi/assay. At the designated time points, duplicate aliquots of vesicle suspension (25 p,g protein) were withdrawn and counted as described in the text. Data are expressed as mean + S.D. of at least three determinations on two individual preparations of plasma membrane. (b) The assay was performed as above except that Nifedipine was administered at a dose of 1.O mgJkg b.w./day for 4 weeks.
390
K E
Contraception
a 4 Wks RECOVERY . AFTER lREAn.iENT 0 NORMAL
1
Fig. 2: Effect of long-term (12-week) Nifedipine treatment on the kinetics of Ca++-uptake by guinea pig spermatozoa1 plasma membrane vesicles. The drug was administered at a dose of (a) 2.5 i&kg b.w./day and (b) 1.0 mg/kg b.w./day for 12 weeks.
CeUuhr calcium content: At 1 mgjkg b.w./day dose of Nifedipine, calcium content remained unaltered but 2.5 mg/kg b.w./day dosage of Nifedipine for 12 weeks resulted in marked enhancement of calcium content without any noticeable recovery (Table II). After short-term treatment, however, increased levels of calcium were restored within 6 weeks of withdrawal of the drug Qeatment (Table II). DISCUSSION The spermatozoa1 plasma membrane Ca++ - transport system and cellular calcium content stimulated by Nifedipine has been described in the present study. In order to study the net calcium transport across the plasma membrane, the Ca” - ATPase activity was also evaluated. A number of reports have clearly demonstrated the in vitro effect of calcium channel blockers on different spermatozoa1 characteristics (6,20). Our data with guinea pig spermatozoa demonstrates the correlation between Nifedipine administration and the net calcium transport across the sperm plasma membrane vesicles. In our assay system, Nifedipine does not exert its inhibitory action on spermatozoa1 Ca*+ build-up which it was otherwise expected to do. Several other studies have also reported that the calcium channel ligands failed to inhibit the acrosome reaction in guinea pig spermatozoa (6) and stimulated the calcium-uptake (20). Coupled with the observation that neither hyperpolarization nor depolarization initiated the acrosome reaction (5), these results suggest that the voltage-sensitive channels probably do not control calcium entry into guinea pig spermatozoa. The negligible calcium uptake observed in the control animals is in consonance with the findings of Coronel and Lardy (21). At least three explanations for the drug-enhanced calcium uptake and Ca++-ATPase activity can be put forward. Firstly, the inhibitory effect of calcium channel blockers on Na+/Ca++ antiporter activity (22) can presumably be discounted. Thus, the blocking of calcium efflux could allow the intracellular calcium concentration to rise above the threshold level.
391
Contraception Table II: Results of Nifedipine administration on different biochemical parameters in guinea pig spermatozoa Ionic calcium (nmoles/mg motein)
Ca*-ATPase (nmoles/min/ mg motein)
Control (Saline) 4-week treatment 4-week recovery 6-week recovery
3.99 f 0.22 3.83 f 0.21 3.72 f 0.75
13.82 f 1.39 15.82 + 0.75 15.82 + 0.79
Group I (I .O mglkg b.w.lday) 4-week treatment 4-week recovery 6-week recovery
4.3 1 +- 0.00 3.21 + 0.25 2.74 + 0.44**
61.03 f 1.78”” 65.99 f 1.82** 52.75 f 2.34”*
Group II 4-week 4-week 6-week
6.16 + 0.21** 4.54 + 0.24* 4.28 + 0.06
108.70 + 4.32** 87.77 f 2.74** 82.30 f 2.08**
Short-term treatment
(2.5 mglkg b.w.lday) treatment recovery recovery
Long-term treatment Control (Saline) 12-week treatment 4-week recovery g-week recovery
3.58 + 0.48 3.80 + 0.23 3.74 r 0.12
13.78 f 0.89 16.84 + 1.28 16.56 f 0.78
Group III (I .O mglkg b.w.lday) 12-week treatment 4-week recovery 6-week recovery
3.37 + 0.29 3.60 + 0.10 3.57 + 0.34
14.79 f 0.04 20.68 + 1.38 41.26 + 1.56**
Group IV (2 5 mglkg b.w ./day) 12-week treatment 4-week recovery 6-week recovery
6.89 + O.OO** 8.20 + 1.06** 6.17 f 0.14**
102.47 f 3.64** 73.25 f 2.65** 38.69 + 1.84**
“pcO.05; **p
