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Effect of prostaglandins on human sperm function in vitro and seminal adenosine triphosphate content*
Vol. 49, No.2, February 1988 Printed in U.S.A.
FERTILITY AND STERILITY Copyright " 1988 The American Fertility Society
Effect of prostaglandins on human sperm function in vitro and seminal adenosine triphosphate content*
Claes Gottlieb, M.D. t Kerstin Svanborg, Ph.D. Peter Eneroth, M.D. Marc Bygdeman, M.D. Department of Obstetrics and Gynecology, Karolinska Hospital, Stockholm, Sweden
The aim of the present study was to evaluate the effect of addition of physiologic amounts of different prostaglandins normally present in semen, on sperm motility, on sperm penetration capacity in cervical mucus in vitro, and on the adenosine triphosphate (ATP) concentration in semen. Semen samples were obtained from volunteers who were attending the fertility outpatient clinic. Sperm motility was measured on a video recorder with a built-in timer, sperm penetration by the Kremer test, and ATP by bioluminescence assay. The addition of 19-hydroxy prostaglandin (PG) E to ejaculates positively stimulated sperm motility and sperm penetration capacity. The opposite effect was observed with 19-hydroxy PGF. PGE 1 , PGE 2 , and PGF2 a had no effect on either parameter, while PGF 1a reduced the sperm motility. The addition of 19-hydroxy PGE to ejaculates increased and the addition of 19-hydroxy PGF reduced semen concentrations of ATP. However, only the last-mentioned effect was statistically significant (P < 0.05). It is suggested that, in particular, 19-hydroxy PGE and 19-hydroxy PGF are important regulators of sperm motility and that the effect may be mediated via effects on the ATP content in the spermatozoa. Fertil Steril 49:322, 1988
Human semen contains large amounts of prostaglandins (PGs) belonging to the 1-, 2-, and 3-series, the 2-series being present in the highest concentrations.+ The total amount of PGs in an ejaculate from fertile men is about 1 mg, 1 with the major compounds being PGE 2 and 19-hydroxy PGE (190H PGE). 3 Semen also contains PGF, 19-0H PGF, their 8~-isomers, 2 • 4 and the recently identified 18,19-dehydro derivatives of PGE 1 and PGE 2 •5
Received May 20, 1987; revised and accepted October 13, 1987. * Supported by the Swedish Medical Research Council project no. 17X-05696. t Reprint requests: Claes Gottlieb, M.D., Department of Obstetrics and Gynecology, Karolinska Hospital, S-104 01 Stockholm, Sweden. :j: Omission of the subscript, e.g., PGE, indicates inclusion of all three PGEs (PGE1o PGE 2 , and PGE3 ).
322
Gottlieb et al. Prostaglandins and sperm function
A correlation between fertility and the seminal content of PGs has been described. 6-S Sperm density and sperm motility seem to be related to the concentration of certain PGs. 9 •10 However, the precise function of individual seminal PGs remains to be established. Previous reports from our group 10 and others regarding the effect of PGE and PGF 11- 14 and 19-0H PGE and 19-0H PGF10·•5 •16 on sperm motility indicate that at least the last two groups of PGs are involved in the physiologic regulation of sperm motility. The aim of the present study was to evaluate the effects of different PGs (PGE, PGF, 19-0H PGE, and 19-0H PGF) on sperm function measured in terms of forward sperm motility and sperm penetration capacity in cervical mucus. In addition, the adenosine triphosphate (ATP) content in the spermatozoa has been determined. Fertility and Sterility
Table 1
Sperm Penetration Score before and after Addition of Various Prostaglandins (PGs) to Semen (Mean and Range)
Compound Amount added (~tg/ml)a
No. experiments Untreated Scored (range)
Treated Score (range)
19-0H PGF2a b
19-0H PGE 2 480 (n = 8)
19-0H PGF'
74 (n
7)
=
52 (n
6)
=
(n
=
20
200 (n = 8)
200 (n = 7)
(n
6)
7)
=
8.9 (8.0-9.0)
7.0 (6.0-8.0)
6.5 (4.0-8.5)
8.4 (7.5-9.0)
7.2 (5.0-8.0)
7.1 (5.0-9.0)
**
**
*
**
NS
NS
NS
NS
5.4 (1.0-8.5)
8.4 (7.5-9.0)
6.8 (4.0-9.0)
7.2 (5.0-9.0)
6.8 (3.5-9.0)
8.1 (7.5-9.0)
4.4 (0-6.0)
This figure represents the median concentration. b The semen samples treated with 19-0H PGF 2a are divided into two groups according to the pretreatment scores. • 19-0H-PGF denotes a mixture of 19-hydroxylated prosta-
MATERIALS AND METHODS
Semen samples were obtained in the laboratory from volunteers and patients attending the fertility outpatient clinic. After liquefaction at room temperature, the semen specimens were divided into aliquots. One of these aliquots was used as a control sample. Physiologic amounts of different PG compounds were added to the remaining aliquots. 1 The possible number of compounds to test each semen sample depended on the volume of the sample1 (Tables 1 to 3). Ten minutes later, 5 #tl of semen from both the control and the treated samples were withdrawn for measurement of the ATP concentration, and at the same time the cervical mucus penetration test was startedP Sperm motility was measured in the control samples and the
3.4 (0-8.0)
glandins isolated from a pool of human semen. d No penetration beyond 10 mm of cervical mucus represents score 0. Maximum score = 9 means that more than 50 spermatozoa were found beyond 20 mm. For details, see Materials and Methods.
treated samples after 10 minutes, and at the end of the penetration test 1 hour later. Adenosine Triphosphate Measurement
The ATP concentration in the spermatozoa was measured as described previously. 18 Trichloroacetic acid (TCA) was used as the extraction agent and a bioluminescence assay was performed. Measurement of Sperm Motility
Ten microliters of semen was introduced into a Makler chamber (Sefi-Medical Instruments, Haifa, Israel) 19 with a grid engraved in the coverslip, which contained 100 squares measuring 0.1 mm2 each. Sperm motility was recorded on a video tape
Sperm Motility Score before and 10 Minutes after Addition of Various Prostaglandins (PGs) to Semen (Mean and Range)
Compound Amount added
19-0H PGF2a b
19-0H PGE 2
(~tg/ml)a
No. experiments
Treated Score (range)
PGE2
6.4 8)
=
PGE 1
5.6 (4.5-6.5)
* P < 0.05; **P < 0.01; NS, not significant.
Untreated Scored (range)
(n
PGF2a
5.2 (3.0-7.0)
a
Table 2
PGF1a
(n
600 = 14)
19-0H PGF'
=
10)
(n
=
7)
(n
=
PGF2a
PGE1
PGE2
20 7)
200 (n = 8)
200 (n = 7)
6.4
51
74 (n
PGF1a
6)
(n
=
9)
(n
=
26 (8-63)
31 (11-42)
57 (49-64)
52 (30-80)
41 (22-64)
49 (35-64)
40 (24-64)
50 (24-72)
**
**
**
**
**
NS
NS
NS
39 (15-81)
19 (5-42)
32 (12-64)
40 (0-80)
33 (12-64)
42 (35-56)
38 (2-64)
50 (24-72)
** P < 0.01; NS, not significant. This figure represents the median concentration. b The semen samples treated with 19-0H PGF2a are divided into two groups according to the pretreatment scores. a
Vol. 49, No.2, February 1988
• 19-0H-PGF denotes a mixture of 19-hydroxylated prostaglandins isolated from a pool of human semen. d Mean amount of forward movement. For details, see Materials and Methods. Gottlieb et al. Prostaglandins and sperm function
323
Table 3 ATP Concentrations (pmol/ml) before and after Addition of Various Prostaglandins (PGs) to Semen (Mean and Range) Compound Amount added
19-0H PGE2
19-0H PGF2a
PGFta
PGE1
PGE 2
74
6.4 (n = 6)
200 (n = 7)
200 (n = 7)
No. experiments
600 (n = 13)
Untreated Concentration (range)
17.6 (6.2-27.5)
21.3 (7.8-47.0)
25.2 (6.4-47.0)
19.3 (7.1-43.3)
22.1 (9.0-48.0)
NSb
**
NS
NS
NS
18.6 (8.1-41.4)
18.6 (7.1-45.0)
24.9 (8.8-45.0)
19.1 (7.5-42.8)
21.8 (10.3-49.0)
(~tg/ml)"
Treated Concentration (range)
(n
= 12)
** p < 0.01.
• This figure represents the median concentration. b NS, not significant.
at 37 times magnification using a video recorder with a timer (Panasonic HG 6200, WJ 810, Tokyo, Japan). During the analysis, 50 spermatozoa were selected at random on the still video picture. The video tape then was run in slow motion and the time needed for each of the 50 spermatozoa to traverse a 0.1 mm 2 square was measured using the time display on the monitor. Sperm motility was expressed as the mean amount of forward movement according to the following equation: 5000 L: time JLmls in which L time represents the total time for the spermatozoa to traverse a square of 0.1 mm2. On the video tape, the tail beat frequency and the head turning frequency also were evaluated. The interassay coefficient of variation for the sperm motility test was 4.1% in six measurements performed on each of three different semen samples. Cervical Mucus Penetration Test
The capacity of the spermatozoa to penetrate cervical mucus was determined by an in vitro testP The spermatozoa were allowed to migrate at 37°C in a 10-JLl capillary tube filled with cervical mucus. The migration distance during 1 hour was measured under the microscope. The outcome of the test was described by a penetration score between 0 and 9 points, where 0 represents no sperm migration beyond 10 mm in the capillary tube, and 9 points represents a sperm migration of 20 mm or more by at least 50 spermatozoa. The intra-assay coefficient of variation for the sperm penetration test was 3.1% and it was based on six assays from each of three semen samples. 324
Gottlieb et al. Prostaglandins and sperm function
Prostaglandins
PGE1o PGE 2, PGF1a, and PGF2a were purchased from Sigma Chemicals (St. Louis, MO). 19-0H PGE2 and 19-0H PGF2a, all as methyl esters, were purchased from the Upjohn Company (Kalamazoo, MI). The free acids of 19-0H PGF2,. were obtained from their methyl esters by hydrolysis with potassium hydroxide (0.05 Min methanol) and then extraction with ethyl acetate. In some experiments, 19-0H PGFs extracted from a pool of human semen were used. 20 Hydrolysis of 19-0H PGE2 methyl ester was performed with coral-derived esterase prepared by the Upjohn Company (Kalamazoo, MI). The method described by Schneider et al. 21 was used with slight modification. Thus, the PG compound disolved in ethanol was added to a water suspension of the enzyme. The resultant mixture was stirred for 20 hours and then diluted with acetone. This mixture was stirred for an additional 45 minutes and filtered through an acidwashed seasand column. After evaporation of the acetone, the residue was purified by thin-layer chromatography (TLC). Blank areas from the TLC plate, corresponding to the location of the 19-0H PG zones, also were extracted and this material was used in control experiments. Statistics
The Wilcoxon paired sign test was used in the statistical calculations. The percentage change in each sample was calculated and the mean value of this change is given in the text and figures. RESULTS
Addition of 19-0H PGE2 resulted in a statistically significant increase (124%) in the capacity of Fertility and Sterility
•te
CHANGE IN SPERM PENETRATION
150
100
50
-50 110H 110H PQE2 PG'i• "" 8
n&13
HIOH PGF n:;S
PG~" PG~. n:::l
n:7
PGE1
PGE2
n.-8
n::7
Figure 1 Sperm penetration capacity after addition of different PG compounds. The bars represent the mean of the percentage difference in each sample before and after treatment. 19-0H PGF denotes a mixture of 19-hydroxylated PGs isolated from pooled human semen.
the spermatozoa to penetrate cervical mucus (Fig. 1, Table 1). Two men (not included in Fig. 1 or Table 1) with a pretreatment penetration score of 0 received, after the addition of 19-0H PGE 2 , sperm penetration scores of 8 and 9, respectively. The mean sperm motility was enhanced by 68% (mean value) when measured 10 minutes after addition of the prostaglandin (Fig. 2, Table 2). The increase in sperm motility was still evident but less marked after 60 minutes (average increase, 48%). Control experiments with extracts from the TLC blank areas revealed no effects on the spermatozoa. The hydrolyzed 19-0H PGF2a and the 19-0H PGFs extracted from human semen caused a significant decrease in sperm penetration capacity (Fig. 1, Table 1). When the semen samples with the least favorable pretreatment score were compared with those showing a better test score, the effect of 19-0H PGF2a was numerically more pronounced in the former group. However, the decrease was significant in both groups (Table 1). Following the addition of 19-0H PGF 2a, the decrease in sperm motility was, on the average, 37%. With 19-0H PGF natural mixture, the corresponding figure was 26%. No attempts were made to elucidate dose-response relations (Fig. 1). The addition of PGFla reduced sperm motility by 21%, while the outcome of the Kremer test was unchanged. Neither PGF 2a, PGE 1 , nor PGE 2 had any effect on sperm motility or sperm penetration capacity (Figs. 1 and 2, Tables 1 and 2). Both PGF 1a and PGF2a altered the sperm motility pattern, as judged by a slow motion playback of the video tape. Vol. 49, No. 2, February 1988
After treatment, the tails seemed stiff and the normal S-shaped tail movement diminished, while the tail beat frequency increased by an average of258% (range, 100% to 340%; n = 15; P < 0.01). The head turning frequency was approximately doubled (mean, 105%; range, 50% to 142%; n = 15; P < 0.01) following addition of PGF 1a or PGF2a. The lateral head movement 16 also was increased, but this change was not statistically significant. The other PGs used did not alter the sperm motility pattern. The ATP content in semen decreased by an average of 13% 10 minutes after the addition of 19-0H PGF 2a. A minor increase (6%) in the ATP concentration in semen was found following the addition of 19-0H PGE 2 , but this change was not significant. No change in seminal ATP was observed after the addition of PGE1o PGE 2 , or PGF2a (Table 3). DISCUSSION
In the present study, the effect of various PGs on sperm motility and on the capacity of the spermatozoa to penetrate cervical mucus in vitro was evaluated. To study the effects of different drugs on sperm motility, an objective method must be used. At least two such procedures have been described: the use of a video tape recorder with a built-in timer, as used in the present study, and time-exposure photomicrography. 16 Both methods allow for measurement of forward velocity of the spermatozoa and evaluation of head and tail movements. The Kremer test for the cervical mucus penetration is an established method and its reliability previously has been evaluated in detait22 Its results depend not only on the activity of the spermatozoa,
•t.
CHANGE IN SPERM MOTILITY
100
50
u w w ;;
10 f----J'-'--,-,----r.---,-,,---,r.--,--,----_;.NS~0
;;
PRETREATMENT LEVEL
-50 ' : : :2
;:~HCII
':::
n•14
n•17
n-8
PGJ\. n-1
PGFzGI n•7
PGE 1 n::8
PGE 2 n:7
Figure 2 Sperm motility after addition of different PG compounds. The bars represent the mean of the percentage difference in each sample before and after treatment. 19-0H PGF denotes a mixture of 19-hydroxylated PGs isolated from pooled human semen. Gottlieb et al.
Prostaglandins and sperm function
325
but also on the quality of the cervical mucus. In the present investigation, the influence of the variation in cervical mucus characteristics was compensated for by using mucus withdrawn from the same woman on one occasion in both control and treated semen sample experiments. Because of the limited amounts of cervical mucus available and differences in the endogenous levels of PGs in individual semen samples, we could not perform dose-response studies. Instead, we carried out the experiments with the addition of various PGs that were within the physiologic range (see Bendvold et aU). Neither PGF 2", PGE 1 , nor PGE 2 in the physiologic doses used changed the sperm penetration capacity or sperm motility. PGF 1" reduced sperm motility, but did not alter the sperm penetration capacity. Cohen et al. 12 have reported a reduction in sperm motility with PGF 2", but they used much higher doses (250 J.Lg) or approximately 20 times the endogenous concentration. Aitken and Kelly 16 also found a decrease in forward velocity of spermatozoa after the addition of 67.5 J.Lg PGF 2"/ml. This dose also is higher than the one used in the present study. In slow motion video playback, the frequency of head turning, the lateral head movement, and tail beat frequency of the spermatozoa were increased following the addition of PGF 1" and PGF 2, . The tails seemed stiff and were straight and not smooth and S-shaped, as in the control samples. Aitken and Kelly16 reported a similar effect on head movement as judged by time exposure photomicrography. It is possible that these effects are due to PG interactions with sperm membranes and/ or a shift in intracellular ion concentrations. The observation that the concentration of 19-0H PGE and 19-0H PGF seems to be of importance for the regulation of sperm motility has been reported previously by Bendvold et al. 10 These authors found a positive correlation between sperm motility and the ratio between the concentrations of 19-0H PGE and 19-0H PGF in semen. 10 Previous preliminary results 23 and the data from the present extended study have shown that the addition of physiologic amounts of 19-0H PGF compounds decreased sperm motility and sperm penetration in cervical mucus. The results of the present study also showed that the addition of 19-0H PGE 2 in physiologic amounts enhanced sperm penetration of cervical mucus. In addition, we have been able to confirm the findings of Aitken and Kelly16 that 19-0H PGE improves sperm motility. Furthermore, these authors 16 have reported 326
Gottlieb et al.
Prostaglandins and sperm function
that the addition of 19-0H PGE increased thecapacity of spermatozoa to penetrate zona-free hamster oozytes. It thus appears that 19-0H PG compounds play a vital role in human reproduction. The fact that prostaglandin 19-hydroxylase has been isolated in extracts of the seminal vesicles, 24 the main production site of all prostaglandins in semen, may be taken as further support for this assumption. The mechanism by which seminal prostaglandins, mainly 19-0H PGE and 19-0H PGF, influence sperm motility is not clear. ATP is the major sperm energy source and a positive correlation between the number of progressively motile spermatozoa and seminal ATP content has been demonstrated. 18•25 The concomitant decrease in both ATP levels and sperm motility following the addition of 19-0H PGF indicate that the effect of the 19-0H PGs on sperm motility is mediated by an effect on the source of energy of the spermatozoa. In conclusion, the results of the present study further support the assumption that 19-0H PGE and 19-0H PGF -in human semen in particularare important regulators of sperm motility and that they influence the sperm penetration of cervical mucus. These results may have important therapeutic implications, as indicated by the dramatic improvement obtained after the addition of 19-0H PGE 2 to the two samples with a sperm penetration score of 0 prior to this treatment. It thus seems justified to initiate clinical studies to evaluate possible beneficial effects of the addition of 19-0H PGE to semen samples used for husband or donor insemination. Acknowledgments. We thank John E. Pike, Ph.D., the Upjohn Company, (Kalamazoo, MI) for his generous gift of the 19-hydroxy prostaglandins. We are grateful to Mrs. Eva Lilliehook, Mrs. Solveig Johansson, Mrs. Astrid Hiiggblad, and Mrs. Eva Andersson for skillful technical assistance, and to Zoe Walsh, M.D., for English language revision.
REFERENCES 1. Bendvold E, Gottlieb C, Svanborg K, Bygdeman M, Ene-
2.
3. 4. 5.
roth P: Concentration of prostaglandins in seminal fluid of fertile men. Int J Androl 10:463, 1987 Samuelsson B: Isolation and identification of prostaglandins from human seminal plasma. J Bioi Chern 238:3229, 1963 Kelly RW: Prostaglandins in semen: their occurrence and possible physiological significance. Int J Androl 1:188, 1978 Taylor PL, Kelly RW: The occurrence of 19-hydroxy F prostaglandins in human semen. FEBS Lett 57:22, 1975 Oliw E, Sprecher H, Hamberg M: Isolation of two novel E
Fertility and Sterility
6.
7.
8. 9.
10.
11.
12.
13.
14.
15.
prostaglandins in human seminal fluid. J Bioi Chern 261:2675, 1986 Hawkins DF: Relevance of prostaglandins to problems of human subfertility. In Prostaglandin Symposium of the Worcester Foundation, Edited by PW Ramwell, JE Shaw. New York, Interscience, 1968, p 1 Bygdeman M, Fredricsson B, Svanborg K, Samuelsson B: The relation between fertility and prostaglandin content of seminal fluid in man. Fertil Steril 21:622, 1970 Collier JG, Flower RJ, Stanton SL: Seminal prostaglandins in infertile men. Fertil Steril 26:868, 1975 Kelly RW, Cooper I, Templeton AA: Reduced prostaglandin levels in the semen of men with very high sperm concentration. J Reprod Fertil 56:195, 1979 Bendvold E, Svanborg K, Eneroth P, Gottlieb C, Bygdeman M: The natural variations in prostaglandin concentration in human seminal fluid and its relation to sperm quality. Fertil Steril 41:743, 1984 Eskin BA, Azarbal S, Sepic R, Slate WG: In vitro responses of the spermatozoa-cervical mucus system treated with prostaglandin F 2a. Obstet Gynecol41:436, 1973 Cohen MS, Colin MJ, Golimbu M, Hotchkiss RS: The effects of prostaglandins on sperm motility. Fertil Steril28:78, 1977 Read MD, Schnieden H: Effects of two methyl xanthine derivatives and four prostaglandins on the motility of spermatozoa from volunteers and oligozoospermatic patients. Int J Androl 1:220, 1978 Schlegel W, Rotermund S, Farber G, Nieschlag E: The influence of prostaglandins on sperm motility. Prostaglandins 21:87,1981 Bygdeman M, Gottlieb C, Svanborg K, Swahn ML: The role of prostaglandins in human reproduction: recent advances. In Prostaglandins and Reproduction, Edited by B Samuelsson, R Paoletti, PW Ramwell. New York, Raven Press, 1987, p 1108
Vol. 49, No.2, February 1988
16. Aitken RJ, Kelly RW: Analysis ofthe direct effect of prostaglandins on human sperm function. J Reprod Fertil 73:139, 1985 17. Kremer 1: A simple sperm penetration test. Int J Fertil 10:209, 1965 18. Gottlieb C, Svanborg K, Eneroth P, Bygdeman M: Adenosine triphosphate in human semen: a study on conditions for a bioluminescence assay. Fertil Steril47:992, 1987 19. Makler A: The improved ten-micrometer chamber for rapid sperm count and motility evaluation. Fertil Steril 33:337, 1980 20. Svanborg K, Bygdeman M, Eneroth P, Bendvold E: Quantification of prostaglandins in human seminal fluid. Prostaglandins 24:363, 1982 21. Schneider WP, Bundy GL, Lincoln FH, Daniels EG, Pike JE: Isolation and chemical conversion of prostaglandins from Plexaura Homomalla: preparation of prostaglandin E 2 , prostaglandin F2 a and their 5,6-trans isomers. J Am Chern Soc 99:1222, 1977 22. Ulstein M: Spermiepenetration i cervixsekret och manlig fertilitet. Thesis, Karolinska Institutet, Stockholm, Sweden, 1972 (in Swedish) 23. Bygdeman M, Bendvold E, Gottlieb C, Svanborg K, Eneroth P: Prostaglandins in human seminal fluid and its relation to fertility. In Prostaglandins, Leukotrienes and Lipoxins, Edited by JM Bailey. Washington, Plenum Publishing, 1985, p 423 24. Oliw E, Fahlstadius P, Hamberg M: Isolation and biosynthesis of 20-hydroxyprostaglandins E 1 and E 2 in ram seminal fluid. J Bioi Chern 261:9216, 1986 25. Comhaire F, Vermeulen L, Ghedira K, Mas J, Irvine S, Callipolitis G: Adenosine triphosphate in human semen: a quantitative estimate of fertilizing potential. Fertil Steril 40:500, 1983
Gottlieb et al. Prostaglandins and sperm function
327
FERTILITY AND STERILITY Copyright " 1988 The American Fertility Society
Effect of prostaglandins on human sperm function in vitro and seminal adenosine triphosphate content*
Claes Gottlieb, M.D. t Kerstin Svanborg, Ph.D. Peter Eneroth, M.D. Marc Bygdeman, M.D. Department of Obstetrics and Gynecology, Karolinska Hospital, Stockholm, Sweden
The aim of the present study was to evaluate the effect of addition of physiologic amounts of different prostaglandins normally present in semen, on sperm motility, on sperm penetration capacity in cervical mucus in vitro, and on the adenosine triphosphate (ATP) concentration in semen. Semen samples were obtained from volunteers who were attending the fertility outpatient clinic. Sperm motility was measured on a video recorder with a built-in timer, sperm penetration by the Kremer test, and ATP by bioluminescence assay. The addition of 19-hydroxy prostaglandin (PG) E to ejaculates positively stimulated sperm motility and sperm penetration capacity. The opposite effect was observed with 19-hydroxy PGF. PGE 1 , PGE 2 , and PGF2 a had no effect on either parameter, while PGF 1a reduced the sperm motility. The addition of 19-hydroxy PGE to ejaculates increased and the addition of 19-hydroxy PGF reduced semen concentrations of ATP. However, only the last-mentioned effect was statistically significant (P < 0.05). It is suggested that, in particular, 19-hydroxy PGE and 19-hydroxy PGF are important regulators of sperm motility and that the effect may be mediated via effects on the ATP content in the spermatozoa. Fertil Steril 49:322, 1988
Human semen contains large amounts of prostaglandins (PGs) belonging to the 1-, 2-, and 3-series, the 2-series being present in the highest concentrations.+ The total amount of PGs in an ejaculate from fertile men is about 1 mg, 1 with the major compounds being PGE 2 and 19-hydroxy PGE (190H PGE). 3 Semen also contains PGF, 19-0H PGF, their 8~-isomers, 2 • 4 and the recently identified 18,19-dehydro derivatives of PGE 1 and PGE 2 •5
Received May 20, 1987; revised and accepted October 13, 1987. * Supported by the Swedish Medical Research Council project no. 17X-05696. t Reprint requests: Claes Gottlieb, M.D., Department of Obstetrics and Gynecology, Karolinska Hospital, S-104 01 Stockholm, Sweden. :j: Omission of the subscript, e.g., PGE, indicates inclusion of all three PGEs (PGE1o PGE 2 , and PGE3 ).
322
Gottlieb et al. Prostaglandins and sperm function
A correlation between fertility and the seminal content of PGs has been described. 6-S Sperm density and sperm motility seem to be related to the concentration of certain PGs. 9 •10 However, the precise function of individual seminal PGs remains to be established. Previous reports from our group 10 and others regarding the effect of PGE and PGF 11- 14 and 19-0H PGE and 19-0H PGF10·•5 •16 on sperm motility indicate that at least the last two groups of PGs are involved in the physiologic regulation of sperm motility. The aim of the present study was to evaluate the effects of different PGs (PGE, PGF, 19-0H PGE, and 19-0H PGF) on sperm function measured in terms of forward sperm motility and sperm penetration capacity in cervical mucus. In addition, the adenosine triphosphate (ATP) content in the spermatozoa has been determined. Fertility and Sterility
Table 1
Sperm Penetration Score before and after Addition of Various Prostaglandins (PGs) to Semen (Mean and Range)
Compound Amount added (~tg/ml)a
No. experiments Untreated Scored (range)
Treated Score (range)
19-0H PGF2a b
19-0H PGE 2 480 (n = 8)
19-0H PGF'
74 (n
7)
=
52 (n
6)
=
(n
=
20
200 (n = 8)
200 (n = 7)
(n
6)
7)
=
8.9 (8.0-9.0)
7.0 (6.0-8.0)
6.5 (4.0-8.5)
8.4 (7.5-9.0)
7.2 (5.0-8.0)
7.1 (5.0-9.0)
**
**
*
**
NS
NS
NS
NS
5.4 (1.0-8.5)
8.4 (7.5-9.0)
6.8 (4.0-9.0)
7.2 (5.0-9.0)
6.8 (3.5-9.0)
8.1 (7.5-9.0)
4.4 (0-6.0)
This figure represents the median concentration. b The semen samples treated with 19-0H PGF 2a are divided into two groups according to the pretreatment scores. • 19-0H-PGF denotes a mixture of 19-hydroxylated prosta-
MATERIALS AND METHODS
Semen samples were obtained in the laboratory from volunteers and patients attending the fertility outpatient clinic. After liquefaction at room temperature, the semen specimens were divided into aliquots. One of these aliquots was used as a control sample. Physiologic amounts of different PG compounds were added to the remaining aliquots. 1 The possible number of compounds to test each semen sample depended on the volume of the sample1 (Tables 1 to 3). Ten minutes later, 5 #tl of semen from both the control and the treated samples were withdrawn for measurement of the ATP concentration, and at the same time the cervical mucus penetration test was startedP Sperm motility was measured in the control samples and the
3.4 (0-8.0)
glandins isolated from a pool of human semen. d No penetration beyond 10 mm of cervical mucus represents score 0. Maximum score = 9 means that more than 50 spermatozoa were found beyond 20 mm. For details, see Materials and Methods.
treated samples after 10 minutes, and at the end of the penetration test 1 hour later. Adenosine Triphosphate Measurement
The ATP concentration in the spermatozoa was measured as described previously. 18 Trichloroacetic acid (TCA) was used as the extraction agent and a bioluminescence assay was performed. Measurement of Sperm Motility
Ten microliters of semen was introduced into a Makler chamber (Sefi-Medical Instruments, Haifa, Israel) 19 with a grid engraved in the coverslip, which contained 100 squares measuring 0.1 mm2 each. Sperm motility was recorded on a video tape
Sperm Motility Score before and 10 Minutes after Addition of Various Prostaglandins (PGs) to Semen (Mean and Range)
Compound Amount added
19-0H PGF2a b
19-0H PGE 2
(~tg/ml)a
No. experiments
Treated Score (range)
PGE2
6.4 8)
=
PGE 1
5.6 (4.5-6.5)
* P < 0.05; **P < 0.01; NS, not significant.
Untreated Scored (range)
(n
PGF2a
5.2 (3.0-7.0)
a
Table 2
PGF1a
(n
600 = 14)
19-0H PGF'
=
10)
(n
=
7)
(n
=
PGF2a
PGE1
PGE2
20 7)
200 (n = 8)
200 (n = 7)
6.4
51
74 (n
PGF1a
6)
(n
=
9)
(n
=
26 (8-63)
31 (11-42)
57 (49-64)
52 (30-80)
41 (22-64)
49 (35-64)
40 (24-64)
50 (24-72)
**
**
**
**
**
NS
NS
NS
39 (15-81)
19 (5-42)
32 (12-64)
40 (0-80)
33 (12-64)
42 (35-56)
38 (2-64)
50 (24-72)
** P < 0.01; NS, not significant. This figure represents the median concentration. b The semen samples treated with 19-0H PGF2a are divided into two groups according to the pretreatment scores. a
Vol. 49, No.2, February 1988
• 19-0H-PGF denotes a mixture of 19-hydroxylated prostaglandins isolated from a pool of human semen. d Mean amount of forward movement. For details, see Materials and Methods. Gottlieb et al. Prostaglandins and sperm function
323
Table 3 ATP Concentrations (pmol/ml) before and after Addition of Various Prostaglandins (PGs) to Semen (Mean and Range) Compound Amount added
19-0H PGE2
19-0H PGF2a
PGFta
PGE1
PGE 2
74
6.4 (n = 6)
200 (n = 7)
200 (n = 7)
No. experiments
600 (n = 13)
Untreated Concentration (range)
17.6 (6.2-27.5)
21.3 (7.8-47.0)
25.2 (6.4-47.0)
19.3 (7.1-43.3)
22.1 (9.0-48.0)
NSb
**
NS
NS
NS
18.6 (8.1-41.4)
18.6 (7.1-45.0)
24.9 (8.8-45.0)
19.1 (7.5-42.8)
21.8 (10.3-49.0)
(~tg/ml)"
Treated Concentration (range)
(n
= 12)
** p < 0.01.
• This figure represents the median concentration. b NS, not significant.
at 37 times magnification using a video recorder with a timer (Panasonic HG 6200, WJ 810, Tokyo, Japan). During the analysis, 50 spermatozoa were selected at random on the still video picture. The video tape then was run in slow motion and the time needed for each of the 50 spermatozoa to traverse a 0.1 mm 2 square was measured using the time display on the monitor. Sperm motility was expressed as the mean amount of forward movement according to the following equation: 5000 L: time JLmls in which L time represents the total time for the spermatozoa to traverse a square of 0.1 mm2. On the video tape, the tail beat frequency and the head turning frequency also were evaluated. The interassay coefficient of variation for the sperm motility test was 4.1% in six measurements performed on each of three different semen samples. Cervical Mucus Penetration Test
The capacity of the spermatozoa to penetrate cervical mucus was determined by an in vitro testP The spermatozoa were allowed to migrate at 37°C in a 10-JLl capillary tube filled with cervical mucus. The migration distance during 1 hour was measured under the microscope. The outcome of the test was described by a penetration score between 0 and 9 points, where 0 represents no sperm migration beyond 10 mm in the capillary tube, and 9 points represents a sperm migration of 20 mm or more by at least 50 spermatozoa. The intra-assay coefficient of variation for the sperm penetration test was 3.1% and it was based on six assays from each of three semen samples. 324
Gottlieb et al. Prostaglandins and sperm function
Prostaglandins
PGE1o PGE 2, PGF1a, and PGF2a were purchased from Sigma Chemicals (St. Louis, MO). 19-0H PGE2 and 19-0H PGF2a, all as methyl esters, were purchased from the Upjohn Company (Kalamazoo, MI). The free acids of 19-0H PGF2,. were obtained from their methyl esters by hydrolysis with potassium hydroxide (0.05 Min methanol) and then extraction with ethyl acetate. In some experiments, 19-0H PGFs extracted from a pool of human semen were used. 20 Hydrolysis of 19-0H PGE2 methyl ester was performed with coral-derived esterase prepared by the Upjohn Company (Kalamazoo, MI). The method described by Schneider et al. 21 was used with slight modification. Thus, the PG compound disolved in ethanol was added to a water suspension of the enzyme. The resultant mixture was stirred for 20 hours and then diluted with acetone. This mixture was stirred for an additional 45 minutes and filtered through an acidwashed seasand column. After evaporation of the acetone, the residue was purified by thin-layer chromatography (TLC). Blank areas from the TLC plate, corresponding to the location of the 19-0H PG zones, also were extracted and this material was used in control experiments. Statistics
The Wilcoxon paired sign test was used in the statistical calculations. The percentage change in each sample was calculated and the mean value of this change is given in the text and figures. RESULTS
Addition of 19-0H PGE2 resulted in a statistically significant increase (124%) in the capacity of Fertility and Sterility
•te
CHANGE IN SPERM PENETRATION
150
100
50
-50 110H 110H PQE2 PG'i• "" 8
n&13
HIOH PGF n:;S
PG~" PG~. n:::l
n:7
PGE1
PGE2
n.-8
n::7
Figure 1 Sperm penetration capacity after addition of different PG compounds. The bars represent the mean of the percentage difference in each sample before and after treatment. 19-0H PGF denotes a mixture of 19-hydroxylated PGs isolated from pooled human semen.
the spermatozoa to penetrate cervical mucus (Fig. 1, Table 1). Two men (not included in Fig. 1 or Table 1) with a pretreatment penetration score of 0 received, after the addition of 19-0H PGE 2 , sperm penetration scores of 8 and 9, respectively. The mean sperm motility was enhanced by 68% (mean value) when measured 10 minutes after addition of the prostaglandin (Fig. 2, Table 2). The increase in sperm motility was still evident but less marked after 60 minutes (average increase, 48%). Control experiments with extracts from the TLC blank areas revealed no effects on the spermatozoa. The hydrolyzed 19-0H PGF2a and the 19-0H PGFs extracted from human semen caused a significant decrease in sperm penetration capacity (Fig. 1, Table 1). When the semen samples with the least favorable pretreatment score were compared with those showing a better test score, the effect of 19-0H PGF2a was numerically more pronounced in the former group. However, the decrease was significant in both groups (Table 1). Following the addition of 19-0H PGF 2a, the decrease in sperm motility was, on the average, 37%. With 19-0H PGF natural mixture, the corresponding figure was 26%. No attempts were made to elucidate dose-response relations (Fig. 1). The addition of PGFla reduced sperm motility by 21%, while the outcome of the Kremer test was unchanged. Neither PGF 2a, PGE 1 , nor PGE 2 had any effect on sperm motility or sperm penetration capacity (Figs. 1 and 2, Tables 1 and 2). Both PGF 1a and PGF2a altered the sperm motility pattern, as judged by a slow motion playback of the video tape. Vol. 49, No. 2, February 1988
After treatment, the tails seemed stiff and the normal S-shaped tail movement diminished, while the tail beat frequency increased by an average of258% (range, 100% to 340%; n = 15; P < 0.01). The head turning frequency was approximately doubled (mean, 105%; range, 50% to 142%; n = 15; P < 0.01) following addition of PGF 1a or PGF2a. The lateral head movement 16 also was increased, but this change was not statistically significant. The other PGs used did not alter the sperm motility pattern. The ATP content in semen decreased by an average of 13% 10 minutes after the addition of 19-0H PGF 2a. A minor increase (6%) in the ATP concentration in semen was found following the addition of 19-0H PGE 2 , but this change was not significant. No change in seminal ATP was observed after the addition of PGE1o PGE 2 , or PGF2a (Table 3). DISCUSSION
In the present study, the effect of various PGs on sperm motility and on the capacity of the spermatozoa to penetrate cervical mucus in vitro was evaluated. To study the effects of different drugs on sperm motility, an objective method must be used. At least two such procedures have been described: the use of a video tape recorder with a built-in timer, as used in the present study, and time-exposure photomicrography. 16 Both methods allow for measurement of forward velocity of the spermatozoa and evaluation of head and tail movements. The Kremer test for the cervical mucus penetration is an established method and its reliability previously has been evaluated in detait22 Its results depend not only on the activity of the spermatozoa,
•t.
CHANGE IN SPERM MOTILITY
100
50
u w w ;;
10 f----J'-'--,-,----r.---,-,,---,r.--,--,----_;.NS~0
;;
PRETREATMENT LEVEL
-50 ' : : :2
;:~HCII
':::
n•14
n•17
n-8
PGJ\. n-1
PGFzGI n•7
PGE 1 n::8
PGE 2 n:7
Figure 2 Sperm motility after addition of different PG compounds. The bars represent the mean of the percentage difference in each sample before and after treatment. 19-0H PGF denotes a mixture of 19-hydroxylated PGs isolated from pooled human semen. Gottlieb et al.
Prostaglandins and sperm function
325
but also on the quality of the cervical mucus. In the present investigation, the influence of the variation in cervical mucus characteristics was compensated for by using mucus withdrawn from the same woman on one occasion in both control and treated semen sample experiments. Because of the limited amounts of cervical mucus available and differences in the endogenous levels of PGs in individual semen samples, we could not perform dose-response studies. Instead, we carried out the experiments with the addition of various PGs that were within the physiologic range (see Bendvold et aU). Neither PGF 2", PGE 1 , nor PGE 2 in the physiologic doses used changed the sperm penetration capacity or sperm motility. PGF 1" reduced sperm motility, but did not alter the sperm penetration capacity. Cohen et al. 12 have reported a reduction in sperm motility with PGF 2", but they used much higher doses (250 J.Lg) or approximately 20 times the endogenous concentration. Aitken and Kelly 16 also found a decrease in forward velocity of spermatozoa after the addition of 67.5 J.Lg PGF 2"/ml. This dose also is higher than the one used in the present study. In slow motion video playback, the frequency of head turning, the lateral head movement, and tail beat frequency of the spermatozoa were increased following the addition of PGF 1" and PGF 2, . The tails seemed stiff and were straight and not smooth and S-shaped, as in the control samples. Aitken and Kelly16 reported a similar effect on head movement as judged by time exposure photomicrography. It is possible that these effects are due to PG interactions with sperm membranes and/ or a shift in intracellular ion concentrations. The observation that the concentration of 19-0H PGE and 19-0H PGF seems to be of importance for the regulation of sperm motility has been reported previously by Bendvold et al. 10 These authors found a positive correlation between sperm motility and the ratio between the concentrations of 19-0H PGE and 19-0H PGF in semen. 10 Previous preliminary results 23 and the data from the present extended study have shown that the addition of physiologic amounts of 19-0H PGF compounds decreased sperm motility and sperm penetration in cervical mucus. The results of the present study also showed that the addition of 19-0H PGE 2 in physiologic amounts enhanced sperm penetration of cervical mucus. In addition, we have been able to confirm the findings of Aitken and Kelly16 that 19-0H PGE improves sperm motility. Furthermore, these authors 16 have reported 326
Gottlieb et al.
Prostaglandins and sperm function
that the addition of 19-0H PGE increased thecapacity of spermatozoa to penetrate zona-free hamster oozytes. It thus appears that 19-0H PG compounds play a vital role in human reproduction. The fact that prostaglandin 19-hydroxylase has been isolated in extracts of the seminal vesicles, 24 the main production site of all prostaglandins in semen, may be taken as further support for this assumption. The mechanism by which seminal prostaglandins, mainly 19-0H PGE and 19-0H PGF, influence sperm motility is not clear. ATP is the major sperm energy source and a positive correlation between the number of progressively motile spermatozoa and seminal ATP content has been demonstrated. 18•25 The concomitant decrease in both ATP levels and sperm motility following the addition of 19-0H PGF indicate that the effect of the 19-0H PGs on sperm motility is mediated by an effect on the source of energy of the spermatozoa. In conclusion, the results of the present study further support the assumption that 19-0H PGE and 19-0H PGF -in human semen in particularare important regulators of sperm motility and that they influence the sperm penetration of cervical mucus. These results may have important therapeutic implications, as indicated by the dramatic improvement obtained after the addition of 19-0H PGE 2 to the two samples with a sperm penetration score of 0 prior to this treatment. It thus seems justified to initiate clinical studies to evaluate possible beneficial effects of the addition of 19-0H PGE to semen samples used for husband or donor insemination. Acknowledgments. We thank John E. Pike, Ph.D., the Upjohn Company, (Kalamazoo, MI) for his generous gift of the 19-hydroxy prostaglandins. We are grateful to Mrs. Eva Lilliehook, Mrs. Solveig Johansson, Mrs. Astrid Hiiggblad, and Mrs. Eva Andersson for skillful technical assistance, and to Zoe Walsh, M.D., for English language revision.
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16. Aitken RJ, Kelly RW: Analysis ofthe direct effect of prostaglandins on human sperm function. J Reprod Fertil 73:139, 1985 17. Kremer 1: A simple sperm penetration test. Int J Fertil 10:209, 1965 18. Gottlieb C, Svanborg K, Eneroth P, Bygdeman M: Adenosine triphosphate in human semen: a study on conditions for a bioluminescence assay. Fertil Steril47:992, 1987 19. Makler A: The improved ten-micrometer chamber for rapid sperm count and motility evaluation. Fertil Steril 33:337, 1980 20. Svanborg K, Bygdeman M, Eneroth P, Bendvold E: Quantification of prostaglandins in human seminal fluid. Prostaglandins 24:363, 1982 21. Schneider WP, Bundy GL, Lincoln FH, Daniels EG, Pike JE: Isolation and chemical conversion of prostaglandins from Plexaura Homomalla: preparation of prostaglandin E 2 , prostaglandin F2 a and their 5,6-trans isomers. J Am Chern Soc 99:1222, 1977 22. Ulstein M: Spermiepenetration i cervixsekret och manlig fertilitet. Thesis, Karolinska Institutet, Stockholm, Sweden, 1972 (in Swedish) 23. Bygdeman M, Bendvold E, Gottlieb C, Svanborg K, Eneroth P: Prostaglandins in human seminal fluid and its relation to fertility. In Prostaglandins, Leukotrienes and Lipoxins, Edited by JM Bailey. Washington, Plenum Publishing, 1985, p 423 24. Oliw E, Fahlstadius P, Hamberg M: Isolation and biosynthesis of 20-hydroxyprostaglandins E 1 and E 2 in ram seminal fluid. J Bioi Chern 261:9216, 1986 25. Comhaire F, Vermeulen L, Ghedira K, Mas J, Irvine S, Callipolitis G: Adenosine triphosphate in human semen: a quantitative estimate of fertilizing potential. Fertil Steril 40:500, 1983
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