FERTILITY AND STERILITY Cpyright 0 1986 The American Fertility Sciety Vl. 45, N.2, February 1986 Printed in U.SA. Inhibitin f sperm mtility and agglutinatin f sperm cells by free fatty acids in whle semen* Israel Siegel, Ph.D. t Alan B. Dudkiewicz, Ph.D. Jan Friberg, M.D., Ph.D. Miguel Suarez, M.D. Nrbert Gleicher, M.D. Divisin f Reprductive Immunlgy, Department f Obstetrics and Gyneclgy, Munt Sinai Hspital Medical Center and Rush Medical Cllege, Chicag, Illinis The effects f a serial dilutin f linleic acid n human spermatza in whle semen was tested n 21 semen samples btained frm 11 nrmal vlunteers. The minimal cncentratin f linleic acid required t stp the mvement f at least 75% f the mving sperm ranged frm 1 t> mg/dl. Fifteen f21 (71%) f the semen samples were inhibited by added free fatty acids (FF A) cncentratins that were less than r clse t the physilgic cncentratin ranges f FF A in bld plasma (1 t 30 mg/dlj. The immbilized sperm ften frmed aggregates similar t thse frmed by the actin f autantibdies against sperm cells. Preliminary studies cnducted n a variety f ther FF A have indicated that leic acid (18/1) was less txic than linleic acid (18/2) and that linlenic acid (18/3) was mre txic than linleic acid. The saturated FFA palmitic acid (16/0) and stearic acid (18/0) at cncentratins up t mg/dl shwed little r n txicity t sperm cells. It is suggested that FFA txicity be included amng physilgic factrs that affect the mtility and spntaneus aggregatin f sperm cells. Fertil SteriI45:273, 1986 Free fatty acids (FFA) are highly txic lipid mlecules. Under cnditins in which their txic prperties are nt sufficiently neutralized, they induce txic changes in red cells, neutrphils, platelets, fibrblasts, heart cells, and brain cells. 1 Our recent studies n the effects f parenteral fat emulsins n the immune adherence phenmenn 2 indicated that FF A liberated frm the fat Received December 3, 1984; revised and accepted Nvember 1, 1985. *Supprted by the Fundatin fr Reprductive Medicine, Inc., Chicag, Illinis. treprint requests: Israel Siegel, Ph.D., Directr, Reprductive Immunlgy Labratry, Munt Sinai Hspital Medical Center, Califrnia Avenue at 15th Street, Chicag, Illinis 60608. Vl. 45, N.2, February 1986 emulsins induce txic mrphlgic changes n red cells bth in vitr and in viv. 2 Additinal studies in ur labratries have demnstrated that the nnsaturated FF A are txic nt nly t red cells but als t tumr cells. 3 We then tested the pssible cyttxic effects f FF A n spermatza. In respnse t the additin f physilgic r lwer-than-physilgic cncentratins f FF A t semen, a dramatic inhibitin f sperm mvement ften ccurred. 4 This article describes the cnditins under which FF A are txic t spermatza in whle semen. MATERIALS AND METHODS We btained 21 semen samples frm 11 nrmal human vlunteers with prven fertility. The se- Siegel et al. Txicity f free fatty acid t sperm cells 273
men samples cntained at least 60 x 10 sperml ml and < 30% abnrmal frms and had > % mtility. All semens were stred at rm temperature and used during the day f ejaculatin. SERUM Human bld was btained frm the arm vein f nrmal vlunteers. The bld was allwed t clt, and then the red cells and serum were separated. The serum was stred at - 70 C until use. FATTY ACIDS Oleic (18/1), linleic (18/2), linlenic (1813), stearic (18/0), and palmitic (1610) acids, all apprximately 99% pure, were btained frm Sigma Chemical Cmpany, St. Luis, MO. SALT SOLUTION All cells were washed, and all fatty acids were diluted in a mdified Hanks' balanced salt slutin,5 except when therwise stated. The salt slutin cntained 1.28 x 10-1 M NaCI, 1 x 10-2 M KCI, 2.87 x 10-3 M K 2 HP0 4, 8.8 x 1O-~ M KH 2 P0 4, and 1.7 x 10-4 M HC!. FREE FATTY ACID TOXICITY ASSAYS Assays f nn saturated FF A txicity n sperm were cnducted in autlgus semen as fllws. Semen was distributed in 12 x 75-mm dispsable glass tubes in O.I-ml prtins, except fr ne tube (tube 1), which cntained 0.2 ml f semen. Tw micrliters f the nnsaturated fatty acids were transferred t the tube.cntaining 0.2 ml f semen. The mixture was shaken fr several secnds in a mechanical shaker t yield a semen fatty acid emulsin cntaining 0 mgldl f the FFA. Then 1f1O ml f this mixture was transferred t a tube that cntained 0.1 ml f semen, yielding a mixture cntaining 0 mgldl f FF A. The mixture was again shaken as described abve. Such serial dilutins were cntinued until the fatty acids were diluted t the desired cncentratins. Alternatively, all dilutins were made with half f the cmpnents listed abve in 1.5-ml plyprpylene Eppendrf Flex Micr Test Tubes (Brinkman Instruments Cmpany, Westbury, NY). Because saturated fatty acids are slid at rm temperature, they were disslved at a 10% cncentratin in ht abslute alchl. Then 2 J.LI f the 10% FF A was transferred t a tube cntaining 0.2 ml f semen, yielding an FF A cncentratin f 274 Siegel et al. Txicity f free fatty acid t sperm cells 10 3. The saturated FFA semen mixtures were then serially diluted exactly as described fr the nnsaturated FF A. Cntrls included semenalchl mixtures with alchl cncentratins similar t thse in the semen and alchl-disslved FF A mixtures. In experiments cmparing the txicity f saturated and unsaturated FF A, bth the saturated and unsaturated FF A were disslved in ht alchl. In several cmparative experiments, the FFA were diluted in seminal plasma instead f semen, as fllws. Semen samples were centrifuged at 0 x g fr 30 minutes and the supernatant seminal plasma separated frm the sedimented sperm cells. The FFA were then serially diluted in the seminal plasma accrding t the prcedures described abve fr the semen. A prtin f the sperm sediment was then added t the FFA-seminal plasma mixtures in quantities that wuld yield a sperm cunt similar t that in the untreated' semen. All assays included cntrl mixtures, which cntained sperm cells but n FF A. Pipette tips were changed fr each serial transfer f the fatty acid mixtures. The mixtures were then transferred t a 37 C incubatr flushed with 5% CO 2 and incubated fr 60 minutes. Then J.LI f the mixtures was transferred t a micrscpic slide and bserved under the phase micrscpe. We cunted, sperm cells at randm t determine the percentage f mving sperm. The fact that serial dilutins f the FFA were made in semen intrduces a ptential surce f errr, because sperm are transferred tgether with the FF A during the serial dilutin prcedures. This culd cause a maximum f % inhibitin f the mving sperm in a serially diluted semen-ffa mixture preceded by a FFA-semen mixture in which all the sperm were nnmtile. T cmpensate fr this and fr cunting errrs, we cnsidered nly inhibitins that stpped> 75% f the mving sperm as statistically significant differences between individual cntrl and FF A treated mixtures (P < 0.001, chi-square analysis). Duplicate serially diluted mixtures indicated a twfld t furfld errr in the minimal txic cncentratin f the FF A. ASCORBIC ACID EFFECTS L-ascrbic acid (Sigma Chemical Cmpany) was disslved in the salt slutin at a 1% cncentratin. The ph f the ascrbic acid salt slutin was then adjusted t 7.2 with additins f IN Fertility and Sterility
Table 1. Minimal Cncentratins f Added Linleic Acids that Immbilized> 75% f the Sperm in Semen Dnr % Mving sperm Minimal Cntrls FFA treated FF A required mg/dl 1 87 0 3.8 2 56 14 3 88 3 7.5 4 86 4 15 5 62 0 15 6 91 17 30 6 77 0 30 6 91 0 60 6 97 10 60 7 81 14 15 8 71 13 30 6 67 11 30 9 88 6 60 9 97 4 10 90 13 12 7 87 6 6 9 79 a 11 95 3 6 91 17 6 19 3 9 81 8 1 a Lwer value nt available. f 21 semen samples (71 %) were inhibited by FF A cncentratins f ~ 30 mg/dl. The cncentratins f FF A required t ablish sperm mvement cmpletely in the semen samples were available fr 16 semen samples. The cncentratins were the same as thse required t inhibit 75% f the mving sperm in 4 f 16 (%), 2 times larger in 6 f 16 (38%), 4 times larger in 4 f 16 (%), 8 times larger in 1 f 16 (6%), and times larger in 1 f 16 (6%) semen samples. AGGLUTINATION OF SPERM CELLS In additin t effects n sperm mtility, the FF A caused agglutinatin f the sperm cells. In general, the nnmtile sperm usually frmed aggregates and the mtile sperm did nt agglutinate. This resulted in a remarkable similarity between the prprtin f mtile and nnmtile sperm and the prprtin f agglutinated and nnagglutinated sperm (Table 2) when cunted in the same micrscpic field. NaOH slutin. Assays t test the effects f ascrbic acid were cnducted as fllws. Semen samples were distributed in 0.09-ml prtins in tubes, except fr ne tube that received 0.18 ml f semen. Then 10 fl.l f 1 % ascrbic acid was added t the 0.09-ml prtins f semen and 20 fl.l f 1% ascrbic acid t the 0.18-ml prtin f semen. Thus, each mixture cntained 5.7 mm f ascrbic acid. Cntrl mixtures cntained similar quantities f semen, except that salt slutin, instead f ascrbic acid, was added. Linleic acid was serially diluted in the semenascrbic acid r semen-salt slutin mixtures exactly as described abve fr the pure semen mixtures. The mixtures were then bserved and assayed as described fr assay prcedures in whle semen. RESULTS INHIBITION OF SPERM MOTILITY The effects flinleic acid n the sperm mtility f 21 different semen specimens are summarized in Table 1. The minimal cncentratin f linleic acid required t stp the mvement f> 75% f the mving sperm varied in different semen samples, ranging frm 1 mg/dl t > mg/dl. Fifteen EFFECTS OF INCUBATION PERIODS In preliminary experiments, we cmpared the effects f different incubatin perids n the FF A txicity titers. Incubatin fsemen-ffa mixtures fr 2 t 3 hurs did nt significantly increase the FF A txicity yer that bserved after 1 hur f incubatin. Hwever, prlnged incubatin f the mixtures ften increased the FF A txicity titers by abut 2 times. Table 2. Effect f Linleic Acid n Sperm Agglutinatin and Mtility Linleic acid cncentratin % Agglutinated a % Nnmtile a mg/dl 0 0 2 1 62 31 94 15 86 86 7.5 68 69 3.75 30 30 1.9 38 38 1.0 7 7 2.5 7 31 0 27 10 aagglutinated sperm and nnmtile sperm were cunted in the same micrscpic fields. Linleic acid was added t whle semen. Vl. 45, N.2, February 1986 Siegel et ai. Txicity f free fatty acid t sperm cells 275
Table 3. Reversibility f Txic Effects f Linleic Acid n Sperm Mtility Linleic acid cncentratin mgldl 12.6 6 Nne Befre remval fffaa 4 55 86 % f mtile sperm After remval fffa b 33 62 80 87 67 aspermatza were expsed t the FF A in pure autlgus semen and incubated in a 37 C incubatr fr 60 minutes. bmixtures were sedimented t remve the semen~fatty acid supernatant. The spermatza were resuspended in physilgic salt slutin cntaining 10% human serum. DILUTION OF FREE FATTY ACIDS IN SEMINAL PLASMA In fur semen samples, linleic acid was diluted in seminal plasma as well as in semen. There were n significant differences in the txicities f the FF A between mixtures diluted in. semen r seminal plasma. REVERSIBILITY OF SPERM INHIBITION T test the reversibility f the FF A effects n sperm mvement, we sedimented the FFA-immbilized sperm cells t remve FFA and resuspended the sperm cells in physilgic salt slutin cntaining 10% human serum. Typical results are illustrated in Table 3. The inhibitin f sperm mvement was irreversible r nly partially reversible when sperm were expsed t txic FF A cncentratins at least 2 t 4 times larger than the minimal txic cncentratins f the FF A. EFFECT OF ASCORBIC ACID T test the effects f ascrbic acid (an antiperxidant) n the txicity f FF A n sperm cells, we expsed sperm cells f fur different semen samples t serial dilutins f linleic acid in the presence r absence f ascrbic acid. Typical results are summarized in Table 4. Ascrbic acid did nt affect the txic effects f linleic acid. TOXIC EFFECTS OF OTHER FREE FATTY ACIDS In preliminary studies, we cmpared the txicities f leic acid (18/1) and linlenic acid. (18/3) t that f linleic acid (18/2). Cmparative results btained frm fur different semen samplesindicated that leic acid (18/1) was Jess inhibitry t 276 Siegel et ai. Txicity f free fatty acid t sperm cells sperm mvement than linleic acid and that linlenic acid (18/3) was mre inhibitry than linleic acid. This is illustrated in Tables 5 and 6. In cntrast t the unsaturated FF A, there were little r n inhibitry effects n sperm mvement by the saturated 16- and 18-carbn FF A (Table 7). DISCUSSION FF A have been knwn t cnstitute the mst. metablically active 6 and txic lipid cmpnents. 7 This study is the first demnstratin f the txic prperties f physilgic FF A n sperm cells; The average FF A cncentratin in human serum, as reprted by different investigatrs, is 0.4 t 0.8 meqll (11.2 t 22.4 mg/dl, assuming a mlecular weight f the FF A f 280) (range, 0.3 t 1.2 meq/l; 8.4 t 33.6 mg/dl).l Thus the cncentratin f added FFA required t inhibit the mvements f mst f the sperm samples in this study was within r less than the physilgic cncentratins f FFA in the bld plasma. The methds we used in this study were designed t detect relatively large effects; such as cmplete cessatin f mvement f mst f the sperm cells r agglutinatin f the sperm cells. Additinal studies. are required t determine the effects f FFA, using mre subtle FFA-induced changes n sperm cells. These may cnsist f changes in the rate f sperm mvement r FF A-induced ultrastructural changes in the sperm cells, which are likely t ccur at FF A cncentratins belw thse reprted in this study.. The significance f txic prperties f physilgic FF A n sperm cells and the fertile state is at present unknwn. T answer this questin, mre infrmatin regarding the nrmal cncentratins f FF A and factrs that neutralize FF A txicity in semen is needed. Table 4. Ascrbic Acid and FFA Txicity Linleic acid mgldl 0 0 2 1 32.5 16. 8.1 4 N ascrbic acid 10 74 97 94 % Sperm mtility Ascrbic acid (5.7mM) 10 69 72 88 95 Fertility and Sterility
Table 5. Effects f Linleic Acid (18/2) and Linlenic Acid (18/3) n Sperm Mtility in Vitr FF A cncentratin mg/dl 12.5 6. 3 Linleic acid (18/2) 15 11 46 54 74 67 % Sperm mtility Linlenic acid (18/3) 2 63 62 Only relatively few studies have been dne t determine FFA cncentratins in either semen r seminal plasma. Sctt et al. 8 measured FF A in semen f a variety f species that did nt include man. The cncentratin f FFA ranged frm 0.1 meq/1 (0.28 mg/di) in rabbits t 0.21 meqll (5.88 mg/di) in rams. Brks et al. 9 reprted an average FF A cncentratin in epididymal plasma f rats f 0.958 ± 0.156 meq/1 (26.8 ± 4.4 mg/di). This was abut 4 times higher than the crrespnding FF A cncentratin in rat bld plasma (0.230 ± 0.057 meq/l; 6.4 ± 1.6 mg/di). Abdel et all fund a FF A cncentratin f 8.97 mg/dl (0.32 meqll) in human semen. This is within the lwer cncentratin ranges f FF A in human plasma. The majr plasma cmpnent that neutralizes fatty acid txicity is albumin.l Thus in the presence f albumin, FFA txicity is a functin f the FFA/albumin rati. The ability f albumin t neutralize FF A txicity in plasma efficiently seems t be limited t abut tw FF A per albumin mlecule.l Fr example, an FF Alalbumin rati f 2 t 3 inhibits chemtaxis f neutrphils by abut %,1 makes platelets mre susceptible t aggregatin,1 and induces txic mrphlgic transfrmatin f human red cells.6 A rati f 3 was demnstrated t depress the cntractility f rat heart preparatins. 1 A rati f4 depresses phagcytsis and bactericidal abilities f neutrphilsl and displaces bund bilirubin frm albumin mlecules. ll A rati f 5 changed enzymatic patterns in perfused heart muscle. 1 A rati f7 t 8 causes heart abnrmalities in patients with mycardial infarctins. 1 We culd find n studies dne t determine the FF A/albumin ratis in semen r seminal plasma. Several authrs, hwever, reprted that the albumin cncentratin in whle semen in men is abut 1% t 2% f the albumin cncentratin in bld plasma.12, 13 It can be calculated frm these Vl. 45, N.2, February 1986 reprts that the FF A/albumin rati in semen is abut, an extrardinary high rati that requires cnfirmatin. Additinal. studies are als required t relate the FF A/albumin ratis t the susceptibility f sperm cells t added FF A.. We suggest that the relatively lw cncentratins f albumin in semen may accunt. in part fr the susceptibility f sperm in semen t FF A. Our results indicate that different semen samples may vary in their susceptibility t the txic effects f FF A. These variatins exceeded the inherent errrs in the prcedures. The variatins were evident nt nly in semen samples f different dnrs, but als in semen samples btained at different times frm the same dnr (dnr 9, Table 1). The factrs that cause this variability are unknwn and require further investigatin. One pssible factr may be the natural variatin in the cncentratin f albumin in semen. Fr example, Hekman and Rumke12 shwed a threefld t furfld difference in the albumin cncentratin between ejaculates f different subjects, which greatly exceeds the natural variatins evident in albumin cncentratins f nrmal serum. Our studies indicate that the txicities f FFA were dependent nt nly nthe cncentratins f FF A but als n the type f FF A in the reactin mixtures. The nnsaturated I8-carbn FF A were mre txic than the saturated 16- t 18-carbn FF A. This is cnsistent with the results f avariety f studies that have indicated that the nnsaturated FF A are generally mre txic t tumr cells3, 14-16 and bacterial7 than saturated fatty acids. In recent studies in ur labratry, we have likewise demnstrated that the 16- t 18-carbn FF A were mre txic t mammalian red cells than the 16- t 18-carbn saturated FFA.6 In additin, differences in txicities t sperm cells were evident between different nnsaturated 18-carbn FF A. The fact that leic acid (18/1) was less Table 6. Effects f Linleic Acid (18/2) and Oleic Acid (18/1) n Sperm Mtility in Vitr Fatty acid cncentratin rrg/dl 12.5 6 3 1.5 Linleic acid (18/2) 8.4 14.5 19.3 45 93.4 % Sperm mtility Oleic acid (18/1) 2 8.2 10 15.6 27.3 38 36 Siegel et at Txicity f free fatty acid t sperm cells 277
Table 7. Effects f Stearic (18/0), Palmitic (16/0), and Linleic Acid (18/2) n Sperm Mtility in Vitr FF A cncentratin mgldl 12.5 6.2 0 Stearic (18/0) 55 53 82 84 73 71 % Sperm mtility Palmitic (16/0) 62 73 67 77 84 Linleic (1812) 0 2 13 31 71 txic t sperm cells than linleic acid (18/2) and linlenic acid (18/3) is cnsistent with ther cmparative studies that have indicated that leic acid (18/1) was the least txic 18-carbn FF A t a variety f tumr cells.14-16 Hamfler et al.18 determined the percentages f different FF A in human semen. The percentage f linleic acid was 6.15 ± 1.88, and that f leic acid was 13.04 ± 1.94. Thus the tw unsaturated FFAcnstituted abut 20% f the ttal FFA in semen. The percentage f linlenic acid (which was the mst txic f the 18-carbn FF A) and the abslute cncentratin f the FF A were nt determined in Hamfler's study. The mechanism thrugh which FF A are txic t cells is still unknwn. FF A are surface-active agents,19 but changes in surface tensin alne cannt cmpletely accunt fr the txicities f the FFA.19 Unsaturated fatty acids are generally highly susceptible t perxidatin. Jnes et al.20 demnstrated that exgenusly applied perxides are pwerfully spermicidal. Nissen and Kreyse121 fund that prly mtile spermatza shwed a relatively high rate f endgenus lipid perxidatin. We therefre cnsidered the pssibility that the txic actin f the linleic acid ccurred thrugh the perxides frmed frm linleic acid. T test this hypthesis, we expsed the FF A sperm mixtures t ascrbic acid, a knwn antiperxidant. If txicity ccurred thrugh perxidatin f linleic acid, it wuld be diminished by the presence f ascrbic acid in the mixture. Our results shwed n decrease in FF A txicity by the presence f ascrbic acid (Table 4). This suggests that the txicity f linleic acid t sperm cells des nt ccur thrugh the perxidatin f linleic acid. FF A levels in viv have been knwn t increase t abve their average value in respnse t a variety f cmmn life cnditins. Fr example, plasma cncentratins f FF A have been knwn t increase up t tw r three times their average values in respnse t ingestin f a fatty meal,22 smking,23 caffeine,24 and stress. It is at present nt knwn hw these fluctuatins f FF A levels affect FFA levels in the semen. Further research is therefre required t determine hw such envirnmental factrs affect the levels f FFA in semen and, thus, sperm mtility. Agglutinatin f sperm cells and reductin in sperm mtility have been cnsidered the hallmarks f the txic actin f autantibdies n sperm cells. Our studies indicate that such effects may be prduced nt nly by antibdies, but als by changes in natural FF A cncentratins. We therefre suggest that alng with antibdies and ther factrs, FFA txicity shuld be cnsidered during clinical evaluatins f spntaneus reductins f sperm mtility and agglutinatins f sperm cells. REFERENCES 1. Spectr AA, Fletcher JE: Transprt f fatty acid. In Disturbance in Lipid and Lipprtein Metablism, Edited by JM Dietschy, AM Gtt, JA Ontk. Bethesda, American Physilgical Sciety, 1978, p 22 2. Siegel I, Liu TL, Zaret P, Gleicher N: Parenteral fat emulsins and immune adherence. The effects f triglycerides n red cell and neutrphil immune adherence in vitr and in viv. JAMA 1:1574, 1984 3. Siegel I, Gleicher N: The selective txicities f nnsaturated fatty acids upn mammary ascites tumr cells. Am J Reprd Immunl (Abstr) 6:74, 1984 4. SiegeU, Dudkiewicz A, Friberg J, Gleicher N: The effects f free fatty acids n human sperm cells. Am J Reprd Immunl (Abstr) 6:59, 1984 5. Martin SP, Green R: Methds fr the study f surviving leukcytes. In Methds in Medical Research, Vl 7, Edited by J Warren. Chicag, Year Bk Medical Publisher, 1958, p 136 6. Siegel I, Gleicher N: Unpublished data 7. Kigshi S, It R: High levels f free fatty acids in lymphid cells with special reference t their cyttxicity. Experientia 29:1408, 1975 8. Sctt TW, White IG, Annisn EF: Fatty acids in semen. Bichem J 78:740, 1961 9. Brks DE, Hamiltn DW, Mallek AH: Carnitine and glycerylphsphrylchline in the reprductive tract fthe male rat. J Reprd Fertil 36:141, 1974 10. Abdel Z, El-Haggar SH, Tawadrus GA, Hamada T, Shawky MA, Amin K: Seminal lipids as energy substrate fr the spermatza. Andrlgia 15:9, 1983 11. Kerner JA, Cassani C, Hurwitz R, Berde CB: Mnitring intravenus fat emulsin in nenates with the fatty acid! serum albumin mlar.ratis. JPEN 5:51, 1981 278 Siegel et ai. Txicity f free fatty acid t sperm cells Fertility and Sterility
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