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Prostaglandins and cyclic-amp in human semen
Prost_aglmdtis
Letiotrienes
PRGSTAGLANDINS Ts.
Malachi,
aa
Medicine 8: 55-62,
AND CYCLIC-AMP
1982
IN HUMAN SEMEN
S. Bichachu and I. Halbrecht
B. Gattegno Research Institute of Human Reproduction and Fetal Development, Hasharon Hospital, Petach Tikva, Israel. (Reprint request to IH) ABSTRACT Normospermic, oligospermic and azoospermic semen samples were analysed for prostaglandins of the E groups and for cyclic AMP. The whole procedure was rapidly performed in a nitrogen atmosphere. Higher levels of total PGEs (PGEs+l9-hydroxy PGEs) were found in normospermic or azoospermic semen. The (135.6?_ v/ml) than in either oligospermic average level of cyclic AMP in normospermic semen (32.0 nmole/ml) did not differ significantly from the test groups, but very low levels were found only in oligo and azoospermic semen. The correlation coefficient between cyclic AMP and total-PGE concentrations was t-0.36. The correlation coefficient between cyclic AMP level and percentage of motile sperm cells was +O. 56 (p< 0.0005). Patients who were treated for two months with a daily dose of 50mg clomiphene citrate exhibited significant elevation of cyclic AMP in their seminal fluid. This finding suggests that cyclic AMP is important for sperm motility and that the clomiphene effect on human semen may be mediated by cyclic AMP. INTRODUCTION ---. The concentration of prostaglandins in seminal fluid is several thousand folds higher than in other body tissues and fluids. Thirteen different prostaglandins had been identified in human semen (l), but later studies suggested that all the seminal prostaglandins of groups A and B are artefacts (2.3.4.). Efforts to find out the role of seminal prostaglandins in fertility have not yet been successful. Seminal fluid is also very rich in cyclic
55
AMP.
There are conflicting
data about the influence of cyclic AMP on sperm cells. Some studies reported a positive effect on cells’ quality (5.6.7. ) whereas others could not confirm it (8.9.10.). The aim of this investigation was to perform the analysis under strict anoxic conditions, to determine the levels of PGEs in human semen in different clinical conditions, to find out whether there is any relationship between the concentrations of seminal prostaglandins and cyclic AMP and to see whether the concentrations of these materials can be related to semen quality. We also investigated the effect of clomiphene on seminal cyclic AMP levels of infertile patients. MATERIALS
AND METHODS
Subjects Males of infertile couples, who came to our institute for clinical consultation were included in this study. None of the subjects had received prior medical treatment for infertility. Semen was delivered by mastrubation and its analyses started at once. Seven infertile patients (see Table 3 for details) received a daily dose of 50 mg clomiphene citrate for two months. Microscopic examination and cyclic AMP analysis were performed before onset of treatment and 7-10 days after its completion. Microscopic
examination
Microscopic examinations were performed by one skilled technician. Spermatozoa1 density was measured by a hemocytometer. Motility was expressed by motility grade and percentage. It was graded from 4 to 0. Grade 4 was given for “excellent motility”, very quick straight forward motion. Grade 3 - good motility, grade 2 - moderate motility, grade 1 poor motility and grade 0 - no forward progress. Analyses
of Prostaglandins
Semen specimens were analysed at once or stored under nitrogen at -2O’C up to four days before analysis. Duplicates of 0. 5 ml semen were analysed, and in cases where insufficient material was available the samples were brought to 0.5 ml with saline. Emphasis was put on rapid processing, and the whole procedured was carried out under nitrogen. Extraction procedures, thin layer chromatography separation, quantitative estimation by spectrophotometric method before and after KOH treatment, and recovery tests were a modification of Bydeman’s method (1). Prostaglandins F were not included in this investigation
56
Cyclic
AMP determination
Freshly delivered semen was thoroughly shaken and duplicate samples of 100 ul were diluted lo-fold with tris-EDTA buffer (containing 0.05 M tris and 0.04 M E$TA, adjusted to pH 7.5). At this stage the samples were stored at -20 C. No change in c’AMP concentration in the diluted samples was found after several months of storage. For protein precipitation the samples were thawed, 2 ml ethanol added, and the precipitates allowed to settle for 5 min at room temperature. After centrifugation the precipitates were washed with 1 ml ethanol-water (2:l). The combined sugernatants were evaporated to dryness under a stream of nitrogen at 50 C. The dry material was dissolved in the tris-EDTA buffer to reconstruct a 400-fold dilution of the original semen specimen. Samples of 50 ul were analysed for c’AMP by protein binding assay (Radiochemical Cent I!e, Amersham. CAMP assay Kit, Code TRK 432). Direct assay of the diluted semen samples, without prior protein precipitation, failed to give reliable results. RESULTS Semeg (106-iOx more than not fit into
samples were divided into 3 groups; Azoospegmic, oligospermic cells/ml) and normospermic (minimum 40x10 cells/ml, of which 50% exhibited motility grade 3 or 4) semen. Specimens which did one of these groups were excluded.
Prostaglandins The results
are summarized
in table 1.
Only traces of A and B prostaglandins were detected. Higher levels of total-PGE (PGEs + 19-hydroxy PGEs) were found in normal semen than in azoospermic specimens (0.02yp~~O. 01 student’s t-test). All the other differences of PGE and 19-hydroxy PGE concentrations between the groups were not significant. in order to test the effect of storage on prostaglandins stability 56 samples were stored at -20’ prior to prostaglandin determination. The storage period varied from 31 to 185 days (mean=55 days) and the percentage of PGBs found in those samples varied from 9% to 84% of the total prostaglandins (meandO%). Corralation coefficient between the numr ber of storage days and percentage of PGB was r= +O. 60 (p
57
Table 1.
Cyclic AMP (nmole/ml) of human semen
Semen samples
oli~_~germic
Normospermic N=16
PGE
19-OH PGE
M
38. 3
42.3
76.1
SD SE
26.4 8.8
16.0 5.3
19. 7 6.6
M
25.1
79.2
51.1
130.3
SD SE
16. 1 3.7
39.0 8.9
19.0 4. 4
41.1 9.4
M
32.0
63.0
72. 6
135.6
SD
19.3
SE __
4.8
23.6 5.9
35. 7 8. 9
* Significantly lower than the normospermic (0. Ol< p< 0.02, student’s t test). Cyclic
( ug/ml) /
Cyclic AMP
-
Azoospermic N==9
and prostaglandins
analyses
total PGEs *118.4 35.3 11. 8
-------
39. 8 9. 9
group,
AMP
Average cyclic AMP level of the normospermic group was 32.0 nmole/ml semen (table 1). No significant difference was found between the control and the test groups. Very low levels of cyclic AMP (below 10 nmole/ml) were found only in oligospermic and azoospermic and were never observed in normospermic semen. Very high concentrations (above 50 nmole/ml) were occasionaly found in all three groups. The correlation coefficient between cyclic AMP levels and percentage of cells with motility grade 3 or 4 was r= +O. 56 (p
58
Table
2.
Correlation @mole/ml)
coefficients between Cyclic AMP levels and prostaglandins in human semen
Prostaglandins
Correlation coefficient
Total PGEs ( ug/ml) / PGEs (,ug/ml)
+O. 36
0.02
0.00
PGEs (per cent of total PGs) The effect
Significance level
0.17
of clomiphene
Following clomiphene citrate treatment there was an improved motility in 3 out of 7 patients. The other 4 did not show any change in semen quality (table 3). Yet an increase in seminal cyclic AMP was found in all the patients. The average increase was 12.1 nmole/ml. This elevation is significant (p 4 0.01) according to Wilcoxon’s test for pair differences. Table 3.
Patient No. 1 2 3 4 5 6 7
Cyclic AMP levels in seminal clomiphene citrate treatment Before clomiphene Semen characteristics
treatment CAMP nmole/ml semen
Azoospermic Oligospermia gravis poor motility Oligospermia gravis poor motility Oligospermia gravis poor motility Moderate oligospermia Necospermia Normospermia poor motility Normospermia poor motility
fluid before
and after
After clomiphene Changes in semen characteristics
treatment CAMP nmole/ml semen
60.1 11.0
No change No change
73.2 20.9
52.5
No change
72.0
21. 7
No change
27.0
22.5
Improved motility
29.1
14.6
Improved
motility
33. 7
25. 8
Improved
motility
36.4
59
DISCUSSION In our procedure of seminal prostaglandin analysis we found only traces of the A and B groupso. PGB appeared during storage, although the semen was stored at -20 C. These findings support the idea that seminal A and B prostaglandins are artefacts (2.3.4. ). The role of seminal prostaglandins is still doubtful. Some studies suggested that they are important in their action in the female tract (11.12.13.14. ), and others showed that seminal PGE is too low in unexplained infertility but otherwise normal semen (15) . We tried to find a connection between results of microscopic analyses and PGEs levels. Our data show that total E-prostaglandins in normospermic semen was significantly higher than in azoospermic semen. No other differences in prostaglandins levels between the groups we examined could be detected. Other investigators published contradictory results: high PGE levels in impaired semen (16), low PGE in infartile patients (13) and low PGE levels at very high sperm counts (1’7). Presumably altered seminal prostaglandins levels express one feature of a complicated syndrom which appears as “impaired fertility”, bearing in mind that seminal prostaglandins might originate from several sources (18). Cyclic AMP levels did not show outstanding differences between the three groups, but very low levels llrere found only in azoospermic and oligospermic semen and never in normal semen. A highly significant correlation coefficient was found between percentage of motile cells and seminal cyclic AMP concentration. The effect of cyclic AMP on spermatozoa is generally accepted (5. ‘7. ) although some investigators denied it (8.9. ). The existance of receptors for cyclic AMP on the outer cell membrane were demonstrated (19), and these enable direct action of cyclic AMP on the spermatozoa1 membrane, which affect sperm respiration and motility by changing the permeability of the membrane (20). Our findings of positive correlation between seminal cylic AMP level and sperm motility supports this idea. It seems that besides the direct action of seminal cyclic AMP on the cells another mode of action exists, namely: cyclic AMP production by the spermatozoa1 adenylate cyclase system. The triggers for this system may be prostaglandins (11). A support for this view was given by Didolkar, who concluded (21) that the enhancement of sperm motility by PGE was caused through the cellular cyclic AMP route and by the detection of prostaglandin receptors on the spermatozoa(22). Clomiphene citrate treatment of infertile patients elevation of seminal cyclic AMP levels. The mehanism improves male fertility has not yet been defined but is the drug stimulates release of gonadotrophic hormones
60
induced a significant whereby clomiphene is believed that (23).
Since FSH and LH increase cyclic AMP in male genital tissues (24.25) one possible pathway for the clomiphene stimulation of seminal cyclicAMP could be via an effect of these hormones. Work is in progress to elucidate the effect of clomiphene in normospermic and vasectomised patients. REFERENCES 1.
Bygdeman M, Svanborg K, Samuelsson B. A method for determination of prostaglandins in human seminal fluid. Clin. Chim. Acta 26: 373, 1969.
2.
Kelly RW, Taylor PL, Hearn JP, Short RV. 19-Hydroxyprostaglandin E as a major component of the semen of primates. Nature 20; 544 l&76.
3.
Templeton AA, Cooper I, Kelley RW. Prostaglandin concentration the semen of fertile men. J. Reprod. Fertil. 52; 147, 1978.
4.
Gerozissis K, Dray F. Radioimmunoassay of prostaglandins semen of fertile men. J. Reprod. Fert. 61: 487, 1981.
5.
Cohen MS, Colin MJ, Golimbu M, Hotchkiss RS. The effect of prostaglandins on sperm motility. Fertil. Steril. 28; ‘78, 1977.
6.
Hommonai ZT, Paz G, Sofer A, Kraicer DF, Hare11 A. Effect of Caffeine on motility, viability, oxygen consumption and glycolitic rate of ejaculated human normokinetic and hypokinetic spermatozoa. Int. J. Fertil. 21; 163, 1976.
7.
Schoenfeld C, Amelar RD, Dubin L. Stimulation of ejaculated spermatozoa by caffeine. Fertil. Steril. 26; 158, 1975.
human
8.
Dougherty KA, Cockett ATK, Urry RL. Caffeine, theophyline human sperm motility. Fertil.Steril. 27; 541, 1976.
and
9.
Beck KJ, Schonhofer PS, Rodermund OE, Dinnendahl V, Peters HD. Lack of relationship between cyclic nucleotide levels and spermatozoal function in human semen. Eertil. Steril. 27; 403, 1976.
in
in the
10. Hammerstedt RH, Hay SR. Effect of incubation temperature on motility and CAMP content of bovine sperm. Arch. Biochem. Biophys. 193; 427, 1980. 11. Kelly RW. Prostaglandins Physiological significance.
in semen; Their Occurrence and possible International J. Androl. 1; 188, 1978.
12. Spilman CH, Bergstrom KK, Forbes AD. Effects of 19-hydroxyprostaglandins on oviductal and uterine motility. Prostaglandins 13; 795, 1977.
61
Letiotrienes
PRGSTAGLANDINS Ts.
Malachi,
aa
Medicine 8: 55-62,
AND CYCLIC-AMP
1982
IN HUMAN SEMEN
S. Bichachu and I. Halbrecht
B. Gattegno Research Institute of Human Reproduction and Fetal Development, Hasharon Hospital, Petach Tikva, Israel. (Reprint request to IH) ABSTRACT Normospermic, oligospermic and azoospermic semen samples were analysed for prostaglandins of the E groups and for cyclic AMP. The whole procedure was rapidly performed in a nitrogen atmosphere. Higher levels of total PGEs (PGEs+l9-hydroxy PGEs) were found in normospermic or azoospermic semen. The (135.6?_ v/ml) than in either oligospermic average level of cyclic AMP in normospermic semen (32.0 nmole/ml) did not differ significantly from the test groups, but very low levels were found only in oligo and azoospermic semen. The correlation coefficient between cyclic AMP and total-PGE concentrations was t-0.36. The correlation coefficient between cyclic AMP level and percentage of motile sperm cells was +O. 56 (p< 0.0005). Patients who were treated for two months with a daily dose of 50mg clomiphene citrate exhibited significant elevation of cyclic AMP in their seminal fluid. This finding suggests that cyclic AMP is important for sperm motility and that the clomiphene effect on human semen may be mediated by cyclic AMP. INTRODUCTION ---. The concentration of prostaglandins in seminal fluid is several thousand folds higher than in other body tissues and fluids. Thirteen different prostaglandins had been identified in human semen (l), but later studies suggested that all the seminal prostaglandins of groups A and B are artefacts (2.3.4.). Efforts to find out the role of seminal prostaglandins in fertility have not yet been successful. Seminal fluid is also very rich in cyclic
55
AMP.
There are conflicting
data about the influence of cyclic AMP on sperm cells. Some studies reported a positive effect on cells’ quality (5.6.7. ) whereas others could not confirm it (8.9.10.). The aim of this investigation was to perform the analysis under strict anoxic conditions, to determine the levels of PGEs in human semen in different clinical conditions, to find out whether there is any relationship between the concentrations of seminal prostaglandins and cyclic AMP and to see whether the concentrations of these materials can be related to semen quality. We also investigated the effect of clomiphene on seminal cyclic AMP levels of infertile patients. MATERIALS
AND METHODS
Subjects Males of infertile couples, who came to our institute for clinical consultation were included in this study. None of the subjects had received prior medical treatment for infertility. Semen was delivered by mastrubation and its analyses started at once. Seven infertile patients (see Table 3 for details) received a daily dose of 50 mg clomiphene citrate for two months. Microscopic examination and cyclic AMP analysis were performed before onset of treatment and 7-10 days after its completion. Microscopic
examination
Microscopic examinations were performed by one skilled technician. Spermatozoa1 density was measured by a hemocytometer. Motility was expressed by motility grade and percentage. It was graded from 4 to 0. Grade 4 was given for “excellent motility”, very quick straight forward motion. Grade 3 - good motility, grade 2 - moderate motility, grade 1 poor motility and grade 0 - no forward progress. Analyses
of Prostaglandins
Semen specimens were analysed at once or stored under nitrogen at -2O’C up to four days before analysis. Duplicates of 0. 5 ml semen were analysed, and in cases where insufficient material was available the samples were brought to 0.5 ml with saline. Emphasis was put on rapid processing, and the whole procedured was carried out under nitrogen. Extraction procedures, thin layer chromatography separation, quantitative estimation by spectrophotometric method before and after KOH treatment, and recovery tests were a modification of Bydeman’s method (1). Prostaglandins F were not included in this investigation
56
Cyclic
AMP determination
Freshly delivered semen was thoroughly shaken and duplicate samples of 100 ul were diluted lo-fold with tris-EDTA buffer (containing 0.05 M tris and 0.04 M E$TA, adjusted to pH 7.5). At this stage the samples were stored at -20 C. No change in c’AMP concentration in the diluted samples was found after several months of storage. For protein precipitation the samples were thawed, 2 ml ethanol added, and the precipitates allowed to settle for 5 min at room temperature. After centrifugation the precipitates were washed with 1 ml ethanol-water (2:l). The combined sugernatants were evaporated to dryness under a stream of nitrogen at 50 C. The dry material was dissolved in the tris-EDTA buffer to reconstruct a 400-fold dilution of the original semen specimen. Samples of 50 ul were analysed for c’AMP by protein binding assay (Radiochemical Cent I!e, Amersham. CAMP assay Kit, Code TRK 432). Direct assay of the diluted semen samples, without prior protein precipitation, failed to give reliable results. RESULTS Semeg (106-iOx more than not fit into
samples were divided into 3 groups; Azoospegmic, oligospermic cells/ml) and normospermic (minimum 40x10 cells/ml, of which 50% exhibited motility grade 3 or 4) semen. Specimens which did one of these groups were excluded.
Prostaglandins The results
are summarized
in table 1.
Only traces of A and B prostaglandins were detected. Higher levels of total-PGE (PGEs + 19-hydroxy PGEs) were found in normal semen than in azoospermic specimens (0.02yp~~O. 01 student’s t-test). All the other differences of PGE and 19-hydroxy PGE concentrations between the groups were not significant. in order to test the effect of storage on prostaglandins stability 56 samples were stored at -20’ prior to prostaglandin determination. The storage period varied from 31 to 185 days (mean=55 days) and the percentage of PGBs found in those samples varied from 9% to 84% of the total prostaglandins (meandO%). Corralation coefficient between the numr ber of storage days and percentage of PGB was r= +O. 60 (p
57
Table 1.
Cyclic AMP (nmole/ml) of human semen
Semen samples
oli~_~germic
Normospermic N=16
PGE
19-OH PGE
M
38. 3
42.3
76.1
SD SE
26.4 8.8
16.0 5.3
19. 7 6.6
M
25.1
79.2
51.1
130.3
SD SE
16. 1 3.7
39.0 8.9
19.0 4. 4
41.1 9.4
M
32.0
63.0
72. 6
135.6
SD
19.3
SE __
4.8
23.6 5.9
35. 7 8. 9
* Significantly lower than the normospermic (0. Ol< p< 0.02, student’s t test). Cyclic
( ug/ml) /
Cyclic AMP
-
Azoospermic N==9
and prostaglandins
analyses
total PGEs *118.4 35.3 11. 8
-------
39. 8 9. 9
group,
AMP
Average cyclic AMP level of the normospermic group was 32.0 nmole/ml semen (table 1). No significant difference was found between the control and the test groups. Very low levels of cyclic AMP (below 10 nmole/ml) were found only in oligospermic and azoospermic and were never observed in normospermic semen. Very high concentrations (above 50 nmole/ml) were occasionaly found in all three groups. The correlation coefficient between cyclic AMP levels and percentage of cells with motility grade 3 or 4 was r= +O. 56 (p
58
Table
2.
Correlation @mole/ml)
coefficients between Cyclic AMP levels and prostaglandins in human semen
Prostaglandins
Correlation coefficient
Total PGEs ( ug/ml) / PGEs (,ug/ml)
+O. 36
0.02
0.00
PGEs (per cent of total PGs) The effect
Significance level
0.17
of clomiphene
Following clomiphene citrate treatment there was an improved motility in 3 out of 7 patients. The other 4 did not show any change in semen quality (table 3). Yet an increase in seminal cyclic AMP was found in all the patients. The average increase was 12.1 nmole/ml. This elevation is significant (p 4 0.01) according to Wilcoxon’s test for pair differences. Table 3.
Patient No. 1 2 3 4 5 6 7
Cyclic AMP levels in seminal clomiphene citrate treatment Before clomiphene Semen characteristics
treatment CAMP nmole/ml semen
Azoospermic Oligospermia gravis poor motility Oligospermia gravis poor motility Oligospermia gravis poor motility Moderate oligospermia Necospermia Normospermia poor motility Normospermia poor motility
fluid before
and after
After clomiphene Changes in semen characteristics
treatment CAMP nmole/ml semen
60.1 11.0
No change No change
73.2 20.9
52.5
No change
72.0
21. 7
No change
27.0
22.5
Improved motility
29.1
14.6
Improved
motility
33. 7
25. 8
Improved
motility
36.4
59
DISCUSSION In our procedure of seminal prostaglandin analysis we found only traces of the A and B groupso. PGB appeared during storage, although the semen was stored at -20 C. These findings support the idea that seminal A and B prostaglandins are artefacts (2.3.4. ). The role of seminal prostaglandins is still doubtful. Some studies suggested that they are important in their action in the female tract (11.12.13.14. ), and others showed that seminal PGE is too low in unexplained infertility but otherwise normal semen (15) . We tried to find a connection between results of microscopic analyses and PGEs levels. Our data show that total E-prostaglandins in normospermic semen was significantly higher than in azoospermic semen. No other differences in prostaglandins levels between the groups we examined could be detected. Other investigators published contradictory results: high PGE levels in impaired semen (16), low PGE in infartile patients (13) and low PGE levels at very high sperm counts (1’7). Presumably altered seminal prostaglandins levels express one feature of a complicated syndrom which appears as “impaired fertility”, bearing in mind that seminal prostaglandins might originate from several sources (18). Cyclic AMP levels did not show outstanding differences between the three groups, but very low levels llrere found only in azoospermic and oligospermic semen and never in normal semen. A highly significant correlation coefficient was found between percentage of motile cells and seminal cyclic AMP concentration. The effect of cyclic AMP on spermatozoa is generally accepted (5. ‘7. ) although some investigators denied it (8.9. ). The existance of receptors for cyclic AMP on the outer cell membrane were demonstrated (19), and these enable direct action of cyclic AMP on the spermatozoa1 membrane, which affect sperm respiration and motility by changing the permeability of the membrane (20). Our findings of positive correlation between seminal cylic AMP level and sperm motility supports this idea. It seems that besides the direct action of seminal cyclic AMP on the cells another mode of action exists, namely: cyclic AMP production by the spermatozoa1 adenylate cyclase system. The triggers for this system may be prostaglandins (11). A support for this view was given by Didolkar, who concluded (21) that the enhancement of sperm motility by PGE was caused through the cellular cyclic AMP route and by the detection of prostaglandin receptors on the spermatozoa(22). Clomiphene citrate treatment of infertile patients elevation of seminal cyclic AMP levels. The mehanism improves male fertility has not yet been defined but is the drug stimulates release of gonadotrophic hormones
60
induced a significant whereby clomiphene is believed that (23).
Since FSH and LH increase cyclic AMP in male genital tissues (24.25) one possible pathway for the clomiphene stimulation of seminal cyclicAMP could be via an effect of these hormones. Work is in progress to elucidate the effect of clomiphene in normospermic and vasectomised patients. REFERENCES 1.
Bygdeman M, Svanborg K, Samuelsson B. A method for determination of prostaglandins in human seminal fluid. Clin. Chim. Acta 26: 373, 1969.
2.
Kelly RW, Taylor PL, Hearn JP, Short RV. 19-Hydroxyprostaglandin E as a major component of the semen of primates. Nature 20; 544 l&76.
3.
Templeton AA, Cooper I, Kelley RW. Prostaglandin concentration the semen of fertile men. J. Reprod. Fertil. 52; 147, 1978.
4.
Gerozissis K, Dray F. Radioimmunoassay of prostaglandins semen of fertile men. J. Reprod. Fert. 61: 487, 1981.
5.
Cohen MS, Colin MJ, Golimbu M, Hotchkiss RS. The effect of prostaglandins on sperm motility. Fertil. Steril. 28; ‘78, 1977.
6.
Hommonai ZT, Paz G, Sofer A, Kraicer DF, Hare11 A. Effect of Caffeine on motility, viability, oxygen consumption and glycolitic rate of ejaculated human normokinetic and hypokinetic spermatozoa. Int. J. Fertil. 21; 163, 1976.
7.
Schoenfeld C, Amelar RD, Dubin L. Stimulation of ejaculated spermatozoa by caffeine. Fertil. Steril. 26; 158, 1975.
human
8.
Dougherty KA, Cockett ATK, Urry RL. Caffeine, theophyline human sperm motility. Fertil.Steril. 27; 541, 1976.
and
9.
Beck KJ, Schonhofer PS, Rodermund OE, Dinnendahl V, Peters HD. Lack of relationship between cyclic nucleotide levels and spermatozoal function in human semen. Eertil. Steril. 27; 403, 1976.
in
in the
10. Hammerstedt RH, Hay SR. Effect of incubation temperature on motility and CAMP content of bovine sperm. Arch. Biochem. Biophys. 193; 427, 1980. 11. Kelly RW. Prostaglandins Physiological significance.
in semen; Their Occurrence and possible International J. Androl. 1; 188, 1978.
12. Spilman CH, Bergstrom KK, Forbes AD. Effects of 19-hydroxyprostaglandins on oviductal and uterine motility. Prostaglandins 13; 795, 1977.
61