THE EFFECTS OF PROSTAGLANDINS ON SPERM MOTILITy*t

Transcription

1 FERTILITY AND STERILITY Copyright 1977 The American Fertility Society Vol. 28, No.1, January 1977 Printed in U.S.A. THE EFFECTS OF PROSTAGLANDINS ON SPERM MOTILITy*t MARC S. COHEN, M.D. MICHAEL J. COLIN, M.D. MmCEA GOLIMBU, M.D. ROBERT S. HOTCHKISS, M.D. Department of Urology and Rheumatology, New York University Medical Center, New York, New York Inflammatory exudates around intrauterine devices were found to produce large concentrations of prostaglandin F2 01. To evaluate the effect of this substance, 100 semen specimens were incubated in concentrations 100 times greater than that found in normal semen. Motility indices were calculated, and significant inhibition of sperm motility was found. The effect of prostaglandin E on sperm motility is also reviewed. The physiology of prostaglandins and their mechanism of action via the cyclic nucleotide system are discussed. The prostaglandins (PGs) constitute one of the. most biologically active compounds ever discovered, and the burgeoning prostaglandin literature since the late 1960s reflects the large amount of interest these compounds have received. Their distribution seems to be widespread, and functional roles in virtually every organ system have been suggested. The term prostaglandin is the generic name for a family of closely related 20-carbon fatty acids containing a five-membered ring. They are commonly described as derivations from the hypothetical structure, prostanoic acid!. 2 (see Fig. 1). More than a dozen compounds are recognized to be members of the principal classes of prostaglandins. Structurally, there are five groups of naturally occurring prostaglandins, the difference being the functional groups which are attributed to the five-membered ring (Fig. 2). These are labeled E, F, A, C, and B (see Fig. 2). A numerical subscript after the letter indicates the degree of unsaturation: PGE 1 has one C-C double bond at position 13, while PGE 2 has two. The alpha and B subscripts for the PGF series designate whether the hydroxyl group at C-9 is on the same side of the molecule as the aliphatic side chain containing the carboxylic acid group (a) or whether it is on the opposite side (B). All of the naturally occurring F prostaglandins have the alpha configuration (e.g., PGF 2(1). A large amount of clinical investigation has linked the action of prostaglandins to various effects on the gastrointestinal tract, cardiovascular system, respiratory system, and central nervous system, plus effects in disease states associated with cancer, platelet function, febrile illness, and inflammatory conditions. 1 The role of prostaglandins in reproduction became one of the primary targets of investigation, especially since these compounds were first extracted from human semen. The term "prostaglandin" was introduced by Von Euler in 1935 to describe vasodepressor, smooth musclestimulating substances which he thought were ~ecreted by the prostate gland. 3 It is now known that human seminal fluid is one of the richest sources of prostaglandins, and 13 such compounds have been detected. It is be- 9 H Accepted August 31, *Presented at the Thirty-Second Annual Meeting of The American Fertility Society, April 5 to 9,1976, Las Vegas, Nev. t American Cystoscope Makers, Inc., Urology Prize Essay of H FIG. 1. Biochemical structure of prostanoic acid.

2 Vol. 28, No.1 o OH ! I OH FIG. 2. Naturally occurring groups of prostaglandins. lieved that the seminal prostaglandins account for most of the smooth muscle-stimulating activity that may contribute to ejaculation.4 The main source is probably seminal vesicle secretion.5 From the Karolinska Institute in Sweden, Bygdemann, Hamberg, and Samuelsson were able to quantitate the concentration of prostaglandins in semen.6 The following concentrations in semen of men with normal fertility were determined: PGE1 and PGE2, 15 to 25 JLglml; PGF2a, 2.2 to 4 JLglml. There is evidence that some cases of male infertility are associated with abnormally low semen concentrations of prostaglandin E.7 Recent work by Collier et al.8 demonstrated a highly significant decrease in PGE concentration in a few infertile patients with no other explanation for their infertility. An attempt to improve the semen quality of these patients by the administration of corn oil, which contains the prostaglandin precursor linoleic acid, or caffeine, which inhibits prostaglandin degradation, proved futile. Animal experiments have attempted to demonstrate an effect of prostaglandins E1 and F ~ on sperm transport as judged by fertilization rate These. results have been variable, but the general view is that fertility has not been affected by increased prostaglandin concentration. Many of the efforts in reproductive physiology indicate roles for prostaglandins in conception, menstruation, induction of labor at term, therapeutic abortion, and fertility control. Regarding fertility control, an intense amount of current research relates to the release ofprostaglandins from patients with intrauterine devices (IUDs). Studies in this area have become important because the mechanism by which IUDs exert their antifertility effects remains unestablished.11 It was first proposed by ChadhurP2 that IUDs release prostaglandins by mildly traumatizing the endometrium. The presence of an IUD is associated with an increased production of prost a glandin F in rabbits, rats, hamsters, mice, and EFFECTS OF PROSTAGLANDINS ON SPERM MOTILITY sheep, in that portion of the endometrium which is in contact with, or in close proximity to, the device. Treatment with indomethacin, an inhibitor of prostaglandin production, has also significantly reduced the concentration of these substances in the endometrial IUD area. 14 In several of these animal studies it has also been shown that the concentration of PGF in uterine venous blood increased by at least 1O-fold A major hypothesis for the antifertility effect of PGF in the presence of an IUD is its luteolytic activity, which has been demonstrated in animal studies In humans, PGF has not definitely been shown to have a lyteolytic effect, although a PGF2a receptor was demonstrated in a human corpus luteum.17 One of the mechanisms by which IUDs increase production of prostaglandins is believed to be uterine distention.18 Recently it was demonstrated that the introduction of an IUD into the endometrial cavity results in an infiltration by neutrophils and macrophages.18 The macrophage has been demonstrated to be a potent source of PG production.19 Bray et al,2o were able to measure concentrations of PGs from macrophages that were adherent to IUDs routinely removed from human patients. This concentration was found to be in the range of 250 JLg of PGF2a, with much smaller concentrations of PGE. This is greater than 100 times the concentration reported in normal seminal plasma. We report here a study in which semen was exposed in vitro to this large concentration of PGF~. MATERIALS AND METHODS Semen specimens for this investigation were selected from 100 men who were being evaluated by routine analyses because of male and/or female fertility problems and were selected if they were not older than 3 hours postejaculation with a sperm count greater than 10 millionlml. The percentage of motile cells was estimated. Motility was graded on a scale from 0 to 4, the latter showing the best quality of progression. In order to examine the results of any change in motility in a more uniform manner, a motility index was calculated for each specimen. These estimates were performed by a trained seminologist (motility index = grade offorward progression x % of motile cells). Specimens were then divided into two equal aliquots, one each for control and experimental samples. To each control were added 5 ml of a modified Ringer's buffer solution (120 mm NaCl, 5 mm KCl, 10 mm KH2 P04, 5 mm 79

3 80 COHEN ET AL. January 1977 TABLE 2. Comparison of Motility Indices for Group 2 MgS0 4 H 2 0, and 1 mm Tris-HCI, at ph 7.2). (80 to 125) Control and Experimental Specimens To each experimental sample, 5 ml of this modified at 15 Minutes and 2 Hours buffer (which in addition contained prostaglandin 15 Minutes 2 Hours F 2" to a final concentration of 250 "glml) were Specimen no. Control Experimental Control Experimental added. These experimental specimens were found to have no change in ph, and a minute amount of ethyl alcohol, which was added to the prepared prostaglandin, was tested separately and was found not to affect sperm motility. All specimens were incubated at 3T C, and 0.05-ml aliquots were initially examined at 0 and 15 minutes and at 1, 2, and 3 hours Since in the initial evaluation all significant changes had occurred by 2 hours, the majority of the specimens were examined at 0 and 15 minutes TABLE 1. Comparison of Motility Indices for Group (>125) Control and Experimental Specimens at 15 Minutes and 2 Hours Minutes 2 Hours Specimen no. Control Experimental Control Experimental and at 2 hours. The difference in control motility index at 0 and 15 minutes was negligible. A double-blind experiment was attempted, since the seminologist who examined all of the speci mens was not informed of the nature of the study or of which specimens were control and which experimental Fifteen of the specimens that demonstrated marked changes in percentage of motility were stained with the eosin-nigrosin live staining technique in order to quantitate these percent ages. This staining technique also enabled us to differentiate between sperm immobilization and death. Several of the specimens were also evaluated by electron microscopy for structural changes The specimens were divided into three groups according to the initial motility index: group 1, specimens with a motility index greater than (Table 1); group 2, 34 specimens with a motility index of 80 to 125 (Table 2); and group 3, with a motility index less than 80 (Table 3).

4 Vol. 28, No.1 EFFECTS OF PROSTAGLANDINS ON SPERM MOTILITY 81 TABLE 3. Comparison of Motility Indices for Group 3 «80) Control and Experimental Specimens at 15 Minutes and 2 Hours Specimen no. 15 Minutes 2 Hours Control Experimental Control Experimental < < < AJl indices were expressed by motility index and later converted to mean ± standard error. After completing the initial experiments with prostaglandin F 2a, 10 more specimens were incubated with cyclic nucleotides in order to test their effect on sperm motility. Equal aliquots of semen were tested at 0 minutes and used as controls. To a separate aliquot of semen was added cyclic guanosine 3' :5' _ monophosphate (cyclic GMP) to a final concentration of 345 x 10-6 mglml. Another motility index was determined at 5 minutes. Cyclic adenosine 3' :5' -monophosphate (cyclic AMP) was added to the same specimen to a final concentration of 329 x 10-3 mglml, plus theophylline to a final concentration of 5 x 10-4 M. Motility indices were again calculated at 15 to 25 minutes. RESULTS Prostaglandin F 2a markedly inhibited the motility of human spermatozoa in the entire sample ofl00 patients, as well as in groups 1 and 2. There was less effect in group 3. If we compare changes in sperm motility for each group at 15 minutes, the average controls for all specimens had a mean motility index of ± 3.5 (mean motility index ± standard error). The motility index of the prostaglandintreated specimens decreased to 82.4 ± 4.8. Differences between the control and PG-treated groups were statistically significant (P < 0.001). At 15 minutes, the mean motility index of group 1 was ± 4.5, and this decreased to ± 5.5 in the experimental group. The control index of group 2 was ± 2.2; this decreased to 64.6 ± 4.6 in the experimental specimens. The changes in both of these groups were statistically significant (P < 0.001). In group 3, the index of the controls was reduced from 44.8 ± 3.8 to 37.5 ± 3.9 in the experimental group. This difference was not statistically significant (P < 0.10) (Table 4). In comparing the percentage change in the mean motility index of each group at 15 minutes, we find that the motility index of all groups changed 26.3%; of group 1, 30.7%; of group 2, 35.4%; and of group 3, 16.3% (Fig. 3). Similar results were found in each group at 2 hours. The mean index of the controls for all specimens at this time was ± 4.3, and the experimental index had decreased to 84.8 ± 5.6. The mean index of group 1 controls was ± 5.9. This decreased to 84.8 ± 5.6 in the experimental group. The control index for group 2 was 92.5 ± 2.8 as compared with 41.0 ± 3.8 for the prostaglandin-treated specimens. Once again, differences observed in these three groups were significant at 2 hours (P < 0.001). Group 3 controls, with an index of 38.0 ± 4.1, demonstrated a more Group No. of specimens TABLE 4. Statistical Analysis of Mean Motility ± Index with Time 15 Minutes Control' PGF... p Control' All groups ± ± 4.8 < ± 4.3 (240-15) (1BO-I0) (240-10) Group ± ± 5.5 < ± 5.9 ( ) (1BO-4O) (240-60) Group ± ± 4.6 < ± 2.8 (120-BO) (120-20) (120-40) Group ± ± 3.9 <0.10& 38.0 ± 4.1 (75-15) (75-10) (70-10) "Values are mean motility indices ± standard error. Ranges are indicated in parentheses. &Not statistically significant. 2 Hours PGF... p 59.2 ± 4.6 (180-<1) < ± 5.6 (180-20) < ± 3.8 (70-10) < ± 3.2 (60-<1) <0.005

5 82 COHEN ET AL. January o ALL GROUPS GROUP 1 GROUP 2 GROUP 3 FIG. 3. Graphic comparison of me an motility indices for control and experimental specimens of each group at 15 minutes. statistically significant change (to 25.8 ± 3.2 [P" < 0.005], compared with the value at 15 minutes (Table 4). In reviewing the percentage change in the mean motility index at 2 hours, we notice that in each group there was a more appreciable change than at 15 minutes. These percentages were: 47.1% for all groups, 41.1% for group 1, 55.7% for group 2, and 32.1% for group 3 (Fig. 4). An attempt to compare changes in the mean motility index only for the prostaglandin-treated specimens at 15 minutes and 2 hours is shown in Figure 5. In all groups the percentage change from 15 minutes to 2 hours was 29%. Group 1 showed a percentage change of 28.5%; group 2, 36.6%; and group 3, 31.2%. This comparison enables us to show that, although the changes in groups 1 and 2 were more significant than those in group 3, the specimens which were affected had similar percentage changes with time in all three groups. The eosin-nigrosin stain was used on 15 specimens that showed marked changes in percentage of motile sperm. This is a technique in which the dead sperm are stained dark and the live or o ALL GROUPS GROUP 1 GROUP 2 GROUP 3 FIG. 4. Graphic comparison of me an motility indices for control and experimental specimens of each group at 2 hours o ALL GROUPS GROOP 1 GROUP 2 GROUP 3 FIG. 5. Graphic comparison of changes in mean motil~ty indices for experimental specimens of each group at 15 mmutes and 2 hours. immobile sperm are stained white (Fig. 6). This enabled us to determine that the effect of PGF 2" was probaj:>ly -cidal rather than merely immobilizing. Electron microscopy was performed on three specimens, but no ultrastructural differences could be ascertained between controls and experimentals. In the test specimens for the cyclic nucleotides, a control mean motility index of 140 was determined. The addition of cyclic GMP caused a marked decrease to 72 at 5 minutes. With the addition of cyclic AMP plus theophylline, the mean motility index increased sharply to 135 at 15 to 25 minutes (Fig. 7). DISCUSSION The results presented in this study clearly demonstrate that PGF2", when present in larger than physiologic concentrations, significantly affects sperm motility at 15 minutes and to a greater extent at 2 hours. The specimens with the poorest quality also seem to be the least affected by the presence of this substance. A third finding is that, regardless of the quality of motility of controls, spermatozoa that are adversely affected will be so affected in a similar manner with time (Fig. 5). The effect of PGF 2" on sperm motility seems to contrast with those stimulatory responses that have been previously reported concerning PGE These results indicate that increased levels of PGE 1 would probably improve the quality of sperm motility. This contrast in action of the El and F prostaglandins seems to be apparent wherever these substances are present together in physiologic concentrations.

6 _I _'I' -_... - Vol. 28, No.1 ~ j.~i-- _1111_'~1 ~_'>I<'.-tItl_III! ~_I~ I --.._ EFFECTs OF PROSTAGLANDINS ON SPERM MOTILITY CONTROl C - GMP **C - AMP + THEDPHYLLI NE *5 Minutes **'5-~ Minutes FIG. 7. Graphic representation of changes in mean motility indices of 10 specimens treated with cyclic nucleotides. FIG. 6. Eosin-nigrosin live staining technique showing live and immobilized sperm staining white (a), and dead sperm staining dark (b). Since large quantities of both PGE and PGF are normal constituents of the human lung, the actions of these prostaglandins in the respiratory system have generated a great deal of interest. Because these compounds exert opposing effects on pulmonary smooth muscle, it has been proposed that an imbalance in their production or metabolism might be the basis for some cases of bronchial asthma. To explain further the opposing mechanisms of PGE 1 and PGF on an intracellular level, there is strong evidence that these substances produce their actions by interacting with the adenyl cyclase-cyclic AMP system. 21 Many of the effects of prostaglandins are believed to be due to stimulation, or lack of stimulation of adenyl cyclase resulting in an increase or slight decrease of cyclic AMP. This effect may serve to increase or decrease the levels of cyclic GMP, because these two systems inversely remain in intracellular balance. An impressive argument fof the mediation of the pharmacologic effects of these substances via cyclic AMP has been summarized by Ramwell and Shaw. 21 In fact, in most adenylate cyclase systems in which prostaglandin synthesis and degradation take place, it is generally accepted that PGE is a strong stimulus for accumulation of cyclic AMP.22 By contrast, PGF either did not alter or was responsible for a slight decrease in the production of this nucleotide. 22 These changes in levels of cyclic AMP may.directly affect concentrations of cyclic GMP which are available to the system. There is much recent evidence to indicate that nucleotide coenzymes play an important part in the control mechanisms which regulate motility and metabolism of spermatozoa. Garbers et a1. 23 suggested that bovine sperm motility is at least partially controlled by cylic AMP and/or cyclic GMP. Tash and Mann24 were further able to

7 84 COHENETAL. demonstrate that the determination of the cyclic AMP content in spermatozoa represents a very accurate and sensitive indicator of sperm activity, particularly in detecting the onset of declining motility. In response to known agents that depress sperm motility or shorten their life-span there was an immediate decrease in the level of cyclic AMP. Confirmatory studies involving spermatozoa of the rhesus monkey and man have demonstrated a significant role for cyclic AMP in the regulation of their oxidative metabolism. Substances which are potent stimulators of cyclic AMP, such as PGE 1 and caffeine, have also been shown to stimulate the kinetic activity and motility of human spermatozoa. 25 Thus, conversely, we propose that substances which decrease or do not affect the levels of cyclic AMP or which increase levels of cyclic GMP in a cell system, such as PGF 2" and sodium fiuoride,24 will markedly inhibit sperm motility when available in large enough concentrations. The fact that large concentrations of cyclic GMP markedly inhibit sperm motility was shown in our test experiments with cyclic nucleotides, as well as a reversal of this inhibition with cyclic AMP (see Fig. 7). In light of our results we can conclude that any factor which will increase the PGF2" concentration in the uterus, fallopian tubes, or ovaries may be detrimental to sperm motility and fertilization. Also, any factor which will cause an increase in PGF 2" in the male ejaculate beyond a critical concentration will have the same adverse effect. Because the effect of PGF 2" on sperm motility did not cause complete sterility, we believe that a combination of factors (luteolysis, uterine distention, etc.) involving both the male and female reproductive systems may be the interrelated cause for the relative infertility imposed by intrauterine devices via the action of prostaglandins. In view of our findings it seems that more investigation into the nature of these metabolically active compounds (prostaglandins) should be encouraged in order to elucidate further the unexplained causes of male and female infertility problems. Acknowledgments. The authors would like acknowledge the assistance and technical expertise of Dr. Cy Schoenfeld and Mrs. Edna Thompson of the New York University Medical Center. REFERENCES January Russell PT, Eberle AJ, Cheng HC: The prostaglandins in clinical medicine. A developing role for the clinical chemist. Clin Chern 21:653, Katz RL, Katz GJ: Prostaglandins-basic and clinical considerations. Anesthesiology 40:471, Von Euler US: tiber die spezifische blutdrucksenkende Substanz des menschlichen Prosta- und Somendlasansekretes. Klin Wochenschr 14:1182, Eliasson R, Risley PL: Potentiated response of isolated seminal vesicles to catecholamines and acetylcholine in the presence of PGE. Acta Physiol Scand 67:253, Mann T: Secretory function of the prostate, seminal vesicle, and other male accessory organs of reproduction. J Reprod Fertil37:179, Bygdeman M: Prostaglandins in human seminal fluid and their correlation to fertility. Int J Fertil 14:228, Hawkins DF: Relevance of prostaglandins to problems of human subfertility. In Prostaglandin Symposium of the Worcester Foundation for Experimental Biology, Edited by PW ;Ramwell, JE Shaw. New York, Inter-science, 1968, p 1 8. Collier JG, Flower RJ, Stanton SL: Seminal prostaglandins in infertile men. Fertil Steril 26:868, Spilman CH, Finn AE, Norland JF: Effect of prostaglandins on sperm transport and fertilization in the rabbit. Prostaglandins 4:57, Lubicz-Nawrocki CM, Saksena SK, Chang MC: The effect of prostaglandins E, and F 2" on the fertilizing ability of hamster spermotozoa. J Reprod Fertil 35:557, Thompson IE: The IUD and prostaglandins: a review ofthe evidence. Obstet GynecoI42:617, Chaudhuri G: Intrauterine device: possible role of prost a glandins. Lancet 1:480, Saksena SK, Lau IF, Castracane VD: Prostaglandin mediated action of IUDs. n. F-Prostaglandins (PGF) in the uterine horn of pregnant rats and hamsters with intrauterine devices. Prostaglandins 5:97, Spilman CH, Duby RT: Prostaglandin mediated luteolytic effect of an intrauterine device in sheep. Prostaglandins 2:159, Kar AB, Goswami A, Kamboj VP, Chowdhury SR: Effect of a foreign body on the response of the uterus of ovariectomized rats to estrogens. Steriods 4:159, Gutknecht GD, Wyngarden LJ, Pharriss BB: The effect of prostaglandin F 2" on ovarian and plasma progesterone levels in the pregnant hamster. Proc Soc Exp BioI Med 136:1151, Powell WS, Hammarstrom S, Samuelsson B, Sjoberg B: Prostaglandin F 2" receptor in human corpora lutea (letter to the editor). Lancet 1:1120, Tatum HJ: Intrauterine contraception. Am J Obstet Gynecol 112:1000, Zurrier RB, Weissmann G, Hoffstein S, Kammerman S, Tai HH: Mechanism of lysosomal enzyme release from human leukocytes. n. Effects of camp and cgmp, autonomic agonists, and agents which affect microtubule function. J Clin Invest 53:297, Bray MA, Gordon D, Morley J: Macrophages on intrauterine contraceptive devices produce prostaglandins. Nature 257:227, Ramwell P, Shaw J (Chairmen): The biological significance of the prostaglandins. Ann NY Acad Sci 180:10, 1971

8 Vol. 28, No.1 EFFECTS OF PROSTAGLANDINS ON SPERM MOTILITY Marsh JM, LeMaire WJ: Cyclic AMP accumulation and steroidogenesis in the human corpus luteum: effect of gonadotropins and prostaglandins. J Clin Endocrinol Metab 38:99, Garbers DL, Lust WD, First NL, Lardy HA: Effects of phosphodiesterase inhibitors and cyclic nucleotides on sperm respiration and motility. Biochemistry 10:1825, Tash JS, Mann FRS: Adenosine 3' :5' -cyclic monophosphate in relation to motility and senescence of spermatozoa. Proc R Soc Lond [BioI] 184:109, Schoenfeld C, Amelar RD, Dubin L: Stimulation of ejaculated human spermatozoa by caffeine. Fertil Steril 26: 158, 1975