Contraception
45:239-248,
EFFECTS OF DI-I-PHLORETIN
W.D.
1992
TEE PROSTANOID RECEPTOR PHOSPHATE, UPON HUMAN
ANTAGONIST, SPERM MOTILITY
Ratnasooriya* and G.A.S. Premakumara Department of Zoology University of Colombo Colombo 3, Sri Lanka
Abstract The inhibitory effects of the prostanoid receptor antagonist, di-4-phloretin phosphate (DPP), upon washed human sperm motility were determined in vitro (up to 60 min Concentrations tested were: of incubation). 0.001, 0.01, 0.1, 0.25, 0.5, 2.5, 5.0 and lO.OmM. All concentrations of investigated caused cessation of sperm DPP movement (percentage motility and average forward velocity) and the antimotility effect was essentially The irreversible. concentrations producing 50% inhibition were: percentage motility - 1.31 + 0.56mM (mean + SEW) and average forward velocity - 1.95 + 0.68mM. The mode of antimotility action multi-faceted. At appears to be high and (1OmM) intermediate (2.5mM) concentrations, changes in fluidity of sperm plasmalemma (hypoosmotic swelling test) and loss of viability (nigrosin-eosin stain technique) constitute the primary means by which motility was disrupted. In contrast, these two parameters of motility remained unaltered at a concentration (0.25mM) of DPP, and the mechanism lower precipitating antimotility the effect involve may mobilization of stored calcium ions and modulation of cyclic AMP levels.
*To whom
reprint
requests
Submitted for publication Accepted for publication
Copyright
Q
1992
should
be made.
December 6, 1991 January 13, 1992
Butterworth-Heinemann
240
Contraception
Introduction drugs suitable as vaginal search new The for In this context, several receptor contraceptives continues. been evaluated as potential vaginal have blockers of However, as yet, the potential contraceptives (1,2). receptor antagonist as vaginal contraceptive prostanoid they been tested although may possess agents not has functional competence of considerable power to suppress semen contains milligram amounts of human sperm; prostaglandins (3), and prostaglandins El and E2 are shown egg penetration (4) and sperm motility to enhance (5) a study was initiated to evaluate antimotility Therefore, antagonist di-4the prostanoid receptor potential of phloretin phosphate Na-salt (DPP) on human sperm. Method Semen
samples
produced by masturbation into samples were Semen specimen containers following 3-5 days of sexual abstinence. These samples were allowed to liquefy at room temperature subjected to analysis using (30°C) for 15-25 min, and All ejaculates use standard established procedures (6). >40 x 10 % concentration had a volume of >1.5 ml, sperm motility of >50% and normal morphology of spermatozoa/ml, seminal The spermatozoa were separated from the >60%. plasma by 3 cycles of centrifugation at 500g for 5 min in modified Krebs-Ringer medium BWW (a 8ml volumes of at a bicarbonate buffer) (7), and were finally resuspended for the concentration of 20 x 106 spermatozoa/ml sperm evaluation of the effects of DPP. Evaluation
of antimotilitv
effects
a gift from Leo Aktiebolaget, DPP (Leo 1235) was It was dissolved in normal saline Sweden. Helsingborg, of 2OmM/ml, and (0.9% NaCl, W/V) to obtain a stock solution Five hundred ul this was used within 6 hr of preparation. of washed spermatozoa in BWW was placed in Falcon tubes and an equal volume of either the vehicle (saline) or DPP was added (final concentration of DPP 0.001, 0.01, 0.1, 0.25, mixed well and the time was 5.0 and lO.OmM), 0.5, 2.5, These suspensions were incubated for 60 recorded (0 min). were transferred (at 0, +5, +10, min, and 10 ul aliquots +15, +30 or f60 min) onto separate clean glass slides (31OC) The percentage and covered with a cover glass (22 x 22mm). motile spermatozoa (6) and the average forward velocity (8) were determined at each concentration and time point under phase contrast optics (x250) using a squared grid and a The concentration of DPP crossed micrometer, respectively. motility and spermatozoa which decreased the percentage
241
Contraception
average forward calculated using
50% of control velocity to (Ec50) probit analysis (N = 11 per treatment).
was
of the reversibility of the antimotility A measure obtained by effect was washing (with medium BWW) the spermatozoa1 sample (N = 6) incubated for +5 and +60 min with 0.25 and lO.OmM of DPP by 3 cycles of centrifugation and reassessing the percentage motile (5OOg, for 5 min), spermatozoa. Viability
of spermatozoa
The viability of spermatozoa of the DPP-treated (0.25, 2.5 and lO.OmM) and control samples (N = 6) was investigated using the nigrosin-eosin technique (6) at 0 and +60 min of incubation. Functional
intesritv
of sperm
olasmalemma
This was assessed by using the hypoosmotic swelling test (9) of DPP-treated (0.25, 2.5 and lO.OmM) and control samples (N = 6) following 0 and +60 min of incubation. Measurement of slutamic oxaloacetic activitv in soermatozoal susoension
transaminase
(GOT)
The GOT activity of sperm suspensions treated with lO.OmM DPP or vehicle was measured (umol/min/l) as described previously in detail (10) using the calorimetric method of Reitman and Frankel as described by Wootton (11). Motilitv
in calcium-free
medium
The percentage motility of spermatozoa of DPP-treated and vehicle-treated (N = 6) (0.25mM) samples was investigated in normal BWW medium and calcium-free BWW medium containing 0.2mM EGTA (12) at 0, +15, +30 and +60 min of incubation. Motility
in the
oresence
of caffeine
The percentage motility of spermatozoa of DPP-treated (Oi25mM) and vehicle-treated (N = 6) was investigated in the presence of 2.5mM caffeine (13) at 0, +30 and +60 min of incubation. Motility
in calcium-free
medium
in the oresence
of caffeine
The percentage motility of the spermatozoa of DPP(0.25mM) treated and vehicle-treated (N = 6) was investigated in calcium-free BWW medium containing 0.2mM EGTA and 2.5mM caffeine at 0, +15, +30 and +60 min of incubation.
242
Contraception
Statistics Results are expressed as mean + SEM. Data were analysed by ANOVA and comparisons between control and test groups were made using Duncan's new multiple range test, except for hypoosmotic swelling, nicrosin-eosin test and GOT assay where Student's t-test was used. Results DPP at relatively low concentrations (0.001, 0.01 and percentage O.lmM) impaired significantly (p
45:239-248,
EFFECTS OF DI-I-PHLORETIN
W.D.
1992
TEE PROSTANOID RECEPTOR PHOSPHATE, UPON HUMAN
ANTAGONIST, SPERM MOTILITY
Ratnasooriya* and G.A.S. Premakumara Department of Zoology University of Colombo Colombo 3, Sri Lanka
Abstract The inhibitory effects of the prostanoid receptor antagonist, di-4-phloretin phosphate (DPP), upon washed human sperm motility were determined in vitro (up to 60 min Concentrations tested were: of incubation). 0.001, 0.01, 0.1, 0.25, 0.5, 2.5, 5.0 and lO.OmM. All concentrations of investigated caused cessation of sperm DPP movement (percentage motility and average forward velocity) and the antimotility effect was essentially The irreversible. concentrations producing 50% inhibition were: percentage motility - 1.31 + 0.56mM (mean + SEW) and average forward velocity - 1.95 + 0.68mM. The mode of antimotility action multi-faceted. At appears to be high and (1OmM) intermediate (2.5mM) concentrations, changes in fluidity of sperm plasmalemma (hypoosmotic swelling test) and loss of viability (nigrosin-eosin stain technique) constitute the primary means by which motility was disrupted. In contrast, these two parameters of motility remained unaltered at a concentration (0.25mM) of DPP, and the mechanism lower precipitating antimotility the effect involve may mobilization of stored calcium ions and modulation of cyclic AMP levels.
*To whom
reprint
requests
Submitted for publication Accepted for publication
Copyright
Q
1992
should
be made.
December 6, 1991 January 13, 1992
Butterworth-Heinemann
240
Contraception
Introduction drugs suitable as vaginal search new The for In this context, several receptor contraceptives continues. been evaluated as potential vaginal have blockers of However, as yet, the potential contraceptives (1,2). receptor antagonist as vaginal contraceptive prostanoid they been tested although may possess agents not has functional competence of considerable power to suppress semen contains milligram amounts of human sperm; prostaglandins (3), and prostaglandins El and E2 are shown egg penetration (4) and sperm motility to enhance (5) a study was initiated to evaluate antimotility Therefore, antagonist di-4the prostanoid receptor potential of phloretin phosphate Na-salt (DPP) on human sperm. Method Semen
samples
produced by masturbation into samples were Semen specimen containers following 3-5 days of sexual abstinence. These samples were allowed to liquefy at room temperature subjected to analysis using (30°C) for 15-25 min, and All ejaculates use standard established procedures (6). >40 x 10 % concentration had a volume of >1.5 ml, sperm motility of >50% and normal morphology of spermatozoa/ml, seminal The spermatozoa were separated from the >60%. plasma by 3 cycles of centrifugation at 500g for 5 min in modified Krebs-Ringer medium BWW (a 8ml volumes of at a bicarbonate buffer) (7), and were finally resuspended for the concentration of 20 x 106 spermatozoa/ml sperm evaluation of the effects of DPP. Evaluation
of antimotilitv
effects
a gift from Leo Aktiebolaget, DPP (Leo 1235) was It was dissolved in normal saline Sweden. Helsingborg, of 2OmM/ml, and (0.9% NaCl, W/V) to obtain a stock solution Five hundred ul this was used within 6 hr of preparation. of washed spermatozoa in BWW was placed in Falcon tubes and an equal volume of either the vehicle (saline) or DPP was added (final concentration of DPP 0.001, 0.01, 0.1, 0.25, mixed well and the time was 5.0 and lO.OmM), 0.5, 2.5, These suspensions were incubated for 60 recorded (0 min). were transferred (at 0, +5, +10, min, and 10 ul aliquots +15, +30 or f60 min) onto separate clean glass slides (31OC) The percentage and covered with a cover glass (22 x 22mm). motile spermatozoa (6) and the average forward velocity (8) were determined at each concentration and time point under phase contrast optics (x250) using a squared grid and a The concentration of DPP crossed micrometer, respectively. motility and spermatozoa which decreased the percentage
241
Contraception
average forward calculated using
50% of control velocity to (Ec50) probit analysis (N = 11 per treatment).
was
of the reversibility of the antimotility A measure obtained by effect was washing (with medium BWW) the spermatozoa1 sample (N = 6) incubated for +5 and +60 min with 0.25 and lO.OmM of DPP by 3 cycles of centrifugation and reassessing the percentage motile (5OOg, for 5 min), spermatozoa. Viability
of spermatozoa
The viability of spermatozoa of the DPP-treated (0.25, 2.5 and lO.OmM) and control samples (N = 6) was investigated using the nigrosin-eosin technique (6) at 0 and +60 min of incubation. Functional
intesritv
of sperm
olasmalemma
This was assessed by using the hypoosmotic swelling test (9) of DPP-treated (0.25, 2.5 and lO.OmM) and control samples (N = 6) following 0 and +60 min of incubation. Measurement of slutamic oxaloacetic activitv in soermatozoal susoension
transaminase
(GOT)
The GOT activity of sperm suspensions treated with lO.OmM DPP or vehicle was measured (umol/min/l) as described previously in detail (10) using the calorimetric method of Reitman and Frankel as described by Wootton (11). Motilitv
in calcium-free
medium
The percentage motility of spermatozoa of DPP-treated and vehicle-treated (N = 6) (0.25mM) samples was investigated in normal BWW medium and calcium-free BWW medium containing 0.2mM EGTA (12) at 0, +15, +30 and +60 min of incubation. Motility
in the
oresence
of caffeine
The percentage motility of spermatozoa of DPP-treated (Oi25mM) and vehicle-treated (N = 6) was investigated in the presence of 2.5mM caffeine (13) at 0, +30 and +60 min of incubation. Motility
in calcium-free
medium
in the oresence
of caffeine
The percentage motility of the spermatozoa of DPP(0.25mM) treated and vehicle-treated (N = 6) was investigated in calcium-free BWW medium containing 0.2mM EGTA and 2.5mM caffeine at 0, +15, +30 and +60 min of incubation.
242
Contraception
Statistics Results are expressed as mean + SEM. Data were analysed by ANOVA and comparisons between control and test groups were made using Duncan's new multiple range test, except for hypoosmotic swelling, nicrosin-eosin test and GOT assay where Student's t-test was used. Results DPP at relatively low concentrations (0.001, 0.01 and percentage O.lmM) impaired significantly (p
