- Email: [email protected]
The effect of bovine seminal plasma on the function and integrity of bovine neutrophils
ELSEVIER
THE EFFECT OF BOVINE SEMINAL PLASMA ON THE FUNCTION AND INTEGRITY OF BOVINE NElJTROPHILS R. 0. Gilbert and M. H. Fales Department of Clinical Sciences College of Veterinary Medicine Cornell University, Ithaca, NY 14853 Received for publication: Accepted:
~a~zuary February
z 7, 1995 1,
1996
ABSTRACT Spermatozoa, by virtue of their high content of polyunsaturated fatty acids and limited antioxidant activity, are highly sensitive to lipid peroxidation. Sources of reactive oxygen species in the female genital tract include neutrophils recruited in response to insemination or those contained within the ejaculate, or even spermatozoa themselves. This study was undertaken to investigate the effect of seminal plasma of bulls on bovine neutrophils. Seminal plasma was removed from ejaculates of 6 healthy bulls by centrifugation. Neutrophils were isolated from jugular venous blood samples obtained from lactating Holstein cows. Seminal plasma was found to decrease measured superoxide anion production by activated neutrophils (P = 0.008) in a concentration-dependent manner over the range tested (0 to 16%). This effect may reflect superoxide scavenging by the seminal plasma or direct inhibition of the neutrophils. It has the potential physiological advantage of protecting spermatozoa from oxidative damage. Seminal plasma was found to reduce homotypic neutrophil aggregation dramatically (P c 0.0001). At a seminal plasma concentration of 0.2% neutrophil aggregation was virtually abolished. Homotypic neutrophil aggregation depends on the CD1 1b/CD1 8 (Mac-l) receptor, which also functions as the C3bi receptor (known as complement receptor-3 or CR3). If this receptor is down-regulated, the ability of ncutrophils to phagocytose complement-opsonized microorganisms would be compromised. Finally, seminal plasma was found to increase neutrophil permeability to trypan blue in a concentration and time dependent fashion (P < O.OOOl), indicating a direct cytotoxic effect. Taken together, these findings indicate a profound inhibition of neutrophil function by bovine seminal plasma. While these mechanisms are likely to protect spermatozoa from oxidative damage or phagocytosis, they also have the potential to diminish dcfensc against pathogenic microorganisms. Key words: bovine, leukocyte, neutrophil,
seminal plasma
Acknowledgments This project was funded, in part, by a grant from the National Association of Animal Breeders. Dr. S. C. Barr provided valuable advice on aggregommetry. Eastern Artificial Insemination Cooperative provided the bull semen.
Theriogenology 46:649-658, 0 1996 by Elsewer Science
1996 Inc.
0093-691x/96/$15.00 PII SOO93-691X(96)00216-6
Theriogenology
650 INTRODUCTION
Several critical functions of spermatozoa such as the acrosome reaction and fertilization depend on membrane fusion events. These events require membrane fluidity. Spermatozoa are afforded this membrane fluidity by the high concentration in their plasmalemma of polyunsaturated fatty acids. The high concentration of unsaturated lipids renders spermatozoa exquisitely sensitive to damage by lipid peroxidation (1,16). One source of reactive oxygen species (superoxide anion, 02-; hydrogen peroxide, H202; hydroxyl radical, OH) is neutrophils recruited to the female tract in response to insemination. This response has been found to be rapid (occurring in less than 15 minutes) and exuberant, and occurs in all species examined to date (8,15,19). Oxidative damage not only has the potential to disrupt membrane fluidity and spermatozoa1 motility but may also destabilize DNA, with potential detrimental effects extending even beyond fertilization (5). A protective effect of seminal plasma against oxidative damage mediated by generation of reactive oxygen species by activated neutrophils would confer a functional advantage on spermatozoa. Such an effect has been reported for seminal plasma of humans and horses (14,2 1,22). Spermatozoa express unique differentiation antigens capable of eliciting immune responses in male and female animals (3). Antispermatozoa antibodies have been implicated as a cause of female infertility. It has been widely recognized that seminal plasma of several species exerts an immunosuppressive effect by inhibiting a range of lymphocyte functions, including the ability of T and B cells to proliferate in response to mitogen or antigen challenge and the ability of NK and cytotoxic T cells to recognize and destroy large T cells (3,23). Less attention has been paid to phagocytic and antigen-presenting cells, although it is known that human seminal plasma inhibits phagocytosis by neutrophils and macrophages and impairs the ability of these cells to recognize target antigens by means of surface cytophilic antibodies (3,22). We report here on experiments designed to examine the effect of seminal plasma from bulls on superoxide anion production by bovine neutrophils, and on homotypic aggregation of bovine neutrophils. Seminal plasma was also examined for a potential cytotoxic effect on bovine neutrophils. Lysis of oocytes, erythrocytes and mixed leukocyte suspensions by bovine seminal plasma has been reported (12,18). MATERIALS
AND METHODS
Ejaculates of normal semen were obtained by use of an artificial vagina from 6 bulls in good health and of established fertility. The bulls were housed in a commercial artificial insemination center. Within 90 min of collection, the semen samples were centrifuged at 3,000 x g for 30 min, and the seminal plasma was separated from the spermatozoa. Fresh seminal plasma was used for Experiment 1. The remaining seminal plasma was then stored at -70 “C for Experiments 2 and 3. Neutrophils were isolated from jugular venous blood samples obtained from healthy, lactating Holstein cows according to a modification of the method of Carlson and Kineko (7).
Theriogenology
651
Briefly, blood was obtained using acid citrate dextrose as anticoagulant and centrifuged at 100 x g for 15 min. The plasma, huffy coat and top half of the erythrocyte column were gently aspirated and discarded. Remaining erythrocytes were removed by flash hypotonic lysis by addition of 20 ml of cold (5 “C), sterile water for 60 sec. Isotonicity was restored by addition of 10 ml of 3 x Hanks Buffered Salt solution (HBSS) without calcium or magnesium. The volume of the mixture was made up to 45 ml with 1 x HBSS and centrifuged at 200 x g for 10 min. The supernatant was discarded, and the washing procedure repeated twice more. Isolated neutrophils were counted and resuspended at 2 x lo6 neutrophils/ml in HBSS with divalent cations. This method routinely yielded cell preparations consisting of more than 90% neutrophils (confirmed by examination of Diff-Quick-stained cytocentrifuge preparations) of greater than 90% viability as determined by trypan blue exclusion. For Experiment 1, neutrophil superoxide anion production was determined by the superoxide dismutase-inhibitable reduction of cytochrome C at 38 “C in the presence of 0, 4, 8 and 16 % seminal plasma with and without addition of phorbol myristate acetate (PMA) to activate the neutrophil oxidative burst (4,lO). The reaction mixture consisted of 1O6 neutrophils, 0.8 mg/ml ferricytochrome C, 0 or 100 rig/ml PMA, in 2.5 ml total volume. The reaction was allowed to proceed at 38 “C for 20 min and was stopped by placing samples on ice for 5 min. After centrifugation the absorbance of the supematant was measured at 550 nm. Production of superoxide anion was calculated according to Beer’s Law, ascribing an extension coefficient of 21.1 r&I-l cm -’ to cytochrome C (17). All samples were tested in duplicate. Seminal plasma from each bull was tested with neutrophils from 2 (different) random cows. In Experiment 2, the effect of seminal plasma on homotypic neutrophil aggregation was determined. Neutrophils were obtained as before, and suspended at 106/m1 with seminal plasma at a concentration of 0.2, 0.75,3.0, 12.0 and 45.0 % in a mixture of HBSS and Tyrode’s solution. One milliliter of this reaction mixture was prewarmed and placed into the reaction chamber of a ChronoLog aggregometer under constant agitation with a magnetic stir bar. After equilibration, aggregation was induced by addition of PMA to yield a final concentration of 50 rig/ml. Aggregation was expressed as the change in chamber impedance (in ohms) in 10 min. In Experiment 3, lo6 neutrophils were suspended with a final concentration of seminal plasma of 0, 2.5, 5.0, 10.0, 20.0 or 40.0 % in HBSS with divalent cations and incubated at 38 C for 60 min. Aliquots from each sample were removed at 0, 10, 20, 30 and 60 min and stained with trypan blue (0.2 %) for 10 min. The cells were then examined microscopically, and the proportion of cells excluding the dye was expressed as a percentage after counting 200 neutrophils. Data from Experiment 1 were examined by two-way analysis of variance, with bull and seminal plasma concentration as the independent variables and superoxide anion production as the dependent variable. When the analysis of variance indicated that statistically significant differences did, in fact, exist, they were further explored and examined for statistical significance by use of Dunnett’s method for multiple comparisons. Data from Experiment 2 were analyzed in
652
Theriogenology
essentially the same way except that one-way analysis of variance was used initially because only 1 independent variable (seminal plasma concentration) was used. For the data from Experiment 3, two-way analysis of variance for repeated measures was used initially; the independent variables were seminal plasma concentration and time. Dose-dependency was examined by determining Pearson’s product moment correlation coefficient between seminal plasma concentration and the independent variable in Experiments 1 and 2, and by multiple linear regression (to determine effect of time and seminal plasma concentration) in Experiment 3. For all measures, differences were considered statistically significant at P < 0.05. Calculations were performed on a microcomputer using commercially available software (Sigma&t, Jandel Scientific, Inc.). RESULTS Seminal plasma significantly diminished measured superoxide anion production by stimulated bovine neutrophils (P = 0.008; Figure 1). Stimulated superoxide anion production decreased from 14.73 + 0.29 (mean + pooled SEM) nmol / lo6 cells / 20 min in the absence of seminal plasma to 10.67 f 0.29, 9.41 f 0.29, and 7.17 + 0.29 nmol / lo6 cells / 20 min in the presence of 4, 8 or 16 % seminal plasma, respectively. Dose-dependency of this response was indicated by the correlation between seminal plasma concentration and superoxide anion production (r = -0.48; P = 0.0006). Dunnett’s test indicated that seminal plasma significantly depressed stimulated superoxide anion at 8 and 16 % (P < 0.05) relative to superoxide anion production in the absence of seminal plasma. There was no difference in unstimulated superoxide anion production by the neutrophils in the presence or absence of seminal plasma, indicating that seminal plasma itself did not stimulate the respiratory burst of neutrophils. The effect of seminal plasma donor was not significant (P = 0.61).
653
Theriogenology
0
4 sefhnal
Figure 1.
8 Flasma
16 (%i
The effect of seminalplasmaon the production of superoxideanion by bovine neutrophilsstimulatedby PMA. (Error barsrepresentstandarddeviation; n=6; P = 0.008.)
In Experiment2 seminalplasmadramaticallyreducedhomotypic neutrophil aggregation asmeasuredby the changein aggregometerchamberimpedanceover 10 min after applicationof the PMA stimulus(P < 0.0001; Figure 2). A concentrationof 0.2 % seminalplasmaeffectively abolishedneutrophilaggregation.This and all higher concentrationswere significantly different from 0 % seminalplasma(Dunnett’s test; P < 0.05). This effect was dose-dependent, with increasingseminalplasmaconcentrationsproducingprogressivelygreaternegative changesin aggregometerchamberimpedance.(Pearson’sproduct momentcorrelationcoefficient r = -0.53; P < 0.001). At higherseminalplasmaconcentrationschamberimpedancedecreasedrather than increasingor staying constant. One explanationfor this observationwould be disruption of neutrophil plasmalemmawith leakage of electrolytes into the extracellular medium, which promptedExperiment3.
654
5 35
4
.E
E z
3 2
2
1
5 iti 5
0
z n. .-E k
-1
-2 -3
f
-4
E .E
-5
Et
s 6
Figure2.
0 (Control)
Seminal
0.2%
Plasma
0.75%
2.94%
11.75%
45%
Concentration
The effect of seminalplasmaon homotypic aggregationof bovine neutrophils after stimulationby PMA. Aggregation is measuredas the changein chamber impedancein ohmsover 10 minutesafter applicationof the stimulus. Data are presentedasthe meanand standarddeviation. (n = 6; P < 0.0000001.)
In Experiment 3 seminalplasmawas found to exert a significant cytotoxic effect on neutrophils,as determinedby their ability to exclude the vital dye trypan blue (Figure 3). The effect wassignificantly dependenton time (P < 0.0001)andconcentrationof seminalplasma(P < 0.0001)with a significant interactionbetweenthesevariables(P < O.OOOl),indicating that the effect of seminalplasmaconcentrationwasdependenton the durationof exposure. All seminal plasmaconcentrationsof 2.5 % or greaterhad a significantly (P < 0.05) detrimentaleffect on neutrophil viability after 20 min (40 % seminalplasma)or 30 min (20, 10, 5, 2.5 % seminal plasma). In neutrophilsnot exposedto seminalplasma,cell dye exclusionremainedabove 95 % for the durationof this experiment,and the influenceof time wasnot significant. Multiple linear regressionconfirmedthat the cytotoxic effect of seminalplasmaon bovine neutrophilsincreased with duration of exposure(P < 0.0001) and increasingseminalplasmaconcentration (P < 0.0001).
Theriogenology
655
120
80
60
40
20
0 0
Seminal
3
Plasma
Concentration
The effect of seminal plasma vital dye, trypan blue. Within each represent times 0, 10, 20, 30 and 60 standard error of the mean. (Effects (P < 0.0001) and time-by-seminal statistically significant.)
(%)
on the ability of bovine neutrophils to exclude the cluster of bars (seminal plasma concentration), bars minutes of incubation. Error bars represent pooled of seminal plasma concentration (P < O.OOOl), time plasma concentration interaction (P < 0.0001) are
DISCUSSION The experiments reported here collectively indicate a profound impairment of neutrophil function by bovine seminal plasma. The diminished cytochrome-c reduction observed in Experiment 1 could be due to free radical scavengers in the seminal plasma, or to impairment of the neutrophil capacity for oxidative metabolism. The presence of antioxidants in seminal plasma, including superoxide dismutase (14), and glutathione peroxidase (13) has been reported. Seminal plasma contains high concentrations of prostaglandins, and prostaglandin E2, in particular, has been found to suppress the oxidative burst of neutrophils and monocytes (28). Other workers have ascribed immunosuppressive activity to unidentified high or low molecular weight substances in seminal plasma (18,20,25,26,29).
656
Theriogenology
Homotypic neutrophil aggregation has been found to depend on expression of the Mac-l glycoprotein (CD1 lb/CD1 8) by neutrophils (6,24). This molecule also serves as the receptor for complement fragment C3bi, known as complement receptor 3 or CR3 (27). Function of this receptor mediates phagocytosis of organisms opsonized by complement. Impairment of aggregation is therefore an indirect indication of reduced or faulty expression of this molecule, and may indicate impaired phagocytic ability of the neutrophils exposed to seminal plasma. Negative changes in chamber impedance prompted speculation that seminal plasma may disturb neutrophil membrane integrity, allowing leakage of electrolytes from the cell. Impairment of cellular aggregation was confirmed to occur in addition to this possibility by microscopic examination of neutrophil suspensions stimulated with PMA in the presence or absence of seminal plasma (results not shown). Experiment 3 was designed to determine whether seminal plasma had a direct effect on neutrophil integrity. Exposure to seminal plasma reduced the ability of neutrophils to exclude the vital dye trypan blue. Results reported here may underestimate damage to neutrophils because, it was our impression that some cells were lysed completely, and were therefore not included in the determination of the percentage of cells able to exclude trypan blue. It has previously been reported that bovine seminal plasma has the ability to lyse erythrocytes and mixed suspensions of leukocytes (18) and oocytes (12). The cumulative results of this experiment indicate a profound impairment by seminal plasma of important indices of neutrophil function. The physiological advantage conferred by this mechanism remains largely speculative, and, although seminal plasma components adhere to sperm, it is not known what concentrations of seminal plasma are achieved in the bovine uterus after natural or artificial insemination. However, it seems very likely that the detrimental effect on neutrophils could well be an important factor in the transmission of venereal infections to female animals as well as the pathogenesis of infectious conditions of the male reproductive tract. Oxidative processes have the potential to damage the spermatozoa membranes by lipid peroxidation (2,11,14,16) but, perhaps more importantly, could also alter spermatozoa DNA (5). Protection of spermatozoa from oxidative damage may be an important function of seminal plasma. Residual amounts of seminal plasma may also serve to protect spermatozoa from premature phagocytosis by antigen presenting cells, which, in turn, may help prevent a humoral response to spermatozoa in the female tract. Although lipid peroxidation may injure spermatozoa cells, suggestions have recently been made that spermatozoa capacitation, hyperactivation and the acrosome reaction may be mediated by free oxygen radicals (2,9). Under this hypothesis, seminal plasma may be important both for protecting spermatozoa from exogenous sources of damaging free radicals, and preventing premature onset of pre-fertilization reactions such as capacitation and the acrosome reaction. The source and identity of the factors in seminal plasma responsible for impairment of neutrophil function are not yet known with certainty, although several suggestions have been made. If, as seems likely, accessory sex glands are responsible for production of these factors,
657
Theriogenology
their presence may be important in the pathogenesis and persistence of infections of these organs. In this regard, infections of the vesicular glands of mature bulls are notoriously persistent and resistant to antimicrobial therapy. REFERENCES 1. Aitken RJ, Buckingham D, Harkiss D. Use of a xanthine oxidase free radical generating system to investigate the cytotoxic effects of reactive oxygen species on human spermatozoa. J Reprod Fertil 1993;97:441-50. 2. Aitken RJ, Clarkson JS, Fischel S. Generation of reactive oxygen species, lipid peroxidation, and human spermatozoa function. Biol Reprod 1989;40: 183-97. 3. Alexander NJ, Anderson DJ. Immunology of semen. Fertil Steril 1987;47:192-205. 4. Babior BM, Kipnes RS, Cumette JT. The production by leukocytes of superoxide, a potential bactericidal agent. J Clin Invest 1973;52:741-4. 5. Bernstein C. Sex as a response to oxidative DNA damage. In: Halliwell B, Arouma 01, (eds) DNA and free radicals. Chichester: Ellis Horwood, 1993; 193-2 10. 6. Bochsler PN, DorC M, Neilsen NR, Slauson DO. A monoclonal antibody-defined adhesion-related antigen on bovine neutrophils is required for neutrophil aggregation. Inflammation 1990;14:499-508. 7. Carlson GP, Kaneko JJ. Isolation of leukocytes from bovine peripheral blood. Proc Sot Exp Biol Med 1973;142:853-6. 8. Clark RA, Klebanoff SJ. Generation of a neutrophil chemotactic agent by spermatozoa: role of complement and regulation by seminal plasma factors. J Immunol 1976; 117: 1378-86. 9. De Lamirande E, Gagnon C. Human spermatozoa hyperactivation and capacitation as parts of an oxidative process. Free Radic Biol Med 1993; 14: 157-66. 10. DorC M, Slauson DO, Neilsen NR. Decreased respiratory burst activity in neonatal bovine neutrophils stimulated by protein kinase C agonists. Am J Vet Res 1991;52:375-SO. 11. Gagnon C, Iwasaki A, De Lamirande E, Kovalski N. Reactive oxygen species and human spermatozoa. Ann NY Acad Sci 1991;637:436-44. 12. Ijaz A, Hunter AG, Ayoub M. Lysis of oocytes by bovine spermatozoa and seminal plasma. J Dairy Sci 1989;72:3273-9. 13. Kantola M, Saaranen M, Vanha-Perttula T. Selenium and glutathione peroxidase in seminal plasma of men and bulls. J Reprod Fertil 1988$X3:785-94. 14. Kobayashi T, Miyazaki T, Natori M, Nozawa S. Protective role of superoxide dismutase in human spermatozoa motility: superoxide dismutase activity and lipid peroxide in human seminal plasma and spermatozoa. Hum Reprod 1991;66:987-91. 15. Kotilainen T, Huhtinen M, Katila T. Sperm-induced leukocytosis in the equine uterus. Theriogenology 1994;41:629-36. 16. Kovalski NN, De Lamirande E, Gagnon C. Reactive oxygen species generated by human neutrophils inhibit spermatozoa motility: protective effect of seminal plasma and scavengers. Fertil Steril 1992;58:809-16. 17. Massey V. The microestimation of succinate and the extinction coefficient of cytochrome-c. Biochem Biophys Acta 1959;34:255-8. 18. Matousek J, Stanek R. Immunosuppressive and leucolytic effect of phospholipid binding protein in bull seminal plasma. Anim Reprod Sci 1993;3 1: l-6.
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Theriogenology
19. Pandya IJ, Cohen J. The leukocytic reaction of the human uterine cervix to spermatozoa. Fertil Stcril 1985;43 :4 17-2 1. 20. Perry ACF, Jones R, Hall L. Studies on a novel, secreted form of superoxide dismutase in rat epididymis. J Reprod Fertil, Abstr Series No. 10, 1993:44-Abstr. 84. 2 1. Scheytt AF, Gilbert RO. Stallion seminal plasma suppresses the oxidative burst of equine neutrophils. Proc Conf Res Workers Anim Dis 1993;74:8 abstr. 22. Schopf RI?, Schramm P, Benes P, Morsches B. Seminal plasma-induced suppression of the respiratory burst of polymorphonuclear leukocytes and monocytes. Andrologia 1984;16:124-8. 23. Skibinski G, Kelly RW, Harkiss D, James K. Immunosuppression by human seminal plasma - extracellular organelles (prostasomes) modulate activity of phagocytic cells. Am J Reprod Immunol 1992;28:97-103. 24. Slauson DO, Skrabalak DS, Neilsen NR, Zwahlen RD. Complement-induced equine neutrophil adhesiveness and aggregation. Vet Path01 1987;24:239-49. 25. Vallely PJ, Sharrard RM, Rees RC. The identification of factors in seminal plasma responsible for suppression of natural killer cell activity. Immunology 1988;63 :45 1-6. 26. Voutto ML, Peluso G, Mancino D, Colomra G, Facchiano A, Ielpo MT, Ravagnan G, Metafora S. Inhibition of interleukin-1 release and activity by the rat seminal vesicle protein SV-IV. J Leukoc Biol 1993;53:214-22. 27. Walport MJ. Complement. In: Roitt IM, Brostoff J, Male DK (eds), Immunology. London: Mosby, 1993;12.1-12.17. 28. Weitzman JB, Wells CE, Wright AH, Clark PA, Law SKA. The gene organisation of the human 2 integrin subunit (CD18). FEBS Lett 1991;294:97-103. 29. Wempe F, Wagner S, V.d.Kammer H, Krauhs E, Scheit KH. Seminalplasmin, the major basic protein of bull seminal plasma, is a secretory protein of the seminal vesicles. Biochim Biophys Acta Gen Subj 1990;1034:260-2.
THE EFFECT OF BOVINE SEMINAL PLASMA ON THE FUNCTION AND INTEGRITY OF BOVINE NElJTROPHILS R. 0. Gilbert and M. H. Fales Department of Clinical Sciences College of Veterinary Medicine Cornell University, Ithaca, NY 14853 Received for publication: Accepted:
~a~zuary February
z 7, 1995 1,
1996
ABSTRACT Spermatozoa, by virtue of their high content of polyunsaturated fatty acids and limited antioxidant activity, are highly sensitive to lipid peroxidation. Sources of reactive oxygen species in the female genital tract include neutrophils recruited in response to insemination or those contained within the ejaculate, or even spermatozoa themselves. This study was undertaken to investigate the effect of seminal plasma of bulls on bovine neutrophils. Seminal plasma was removed from ejaculates of 6 healthy bulls by centrifugation. Neutrophils were isolated from jugular venous blood samples obtained from lactating Holstein cows. Seminal plasma was found to decrease measured superoxide anion production by activated neutrophils (P = 0.008) in a concentration-dependent manner over the range tested (0 to 16%). This effect may reflect superoxide scavenging by the seminal plasma or direct inhibition of the neutrophils. It has the potential physiological advantage of protecting spermatozoa from oxidative damage. Seminal plasma was found to reduce homotypic neutrophil aggregation dramatically (P c 0.0001). At a seminal plasma concentration of 0.2% neutrophil aggregation was virtually abolished. Homotypic neutrophil aggregation depends on the CD1 1b/CD1 8 (Mac-l) receptor, which also functions as the C3bi receptor (known as complement receptor-3 or CR3). If this receptor is down-regulated, the ability of ncutrophils to phagocytose complement-opsonized microorganisms would be compromised. Finally, seminal plasma was found to increase neutrophil permeability to trypan blue in a concentration and time dependent fashion (P < O.OOOl), indicating a direct cytotoxic effect. Taken together, these findings indicate a profound inhibition of neutrophil function by bovine seminal plasma. While these mechanisms are likely to protect spermatozoa from oxidative damage or phagocytosis, they also have the potential to diminish dcfensc against pathogenic microorganisms. Key words: bovine, leukocyte, neutrophil,
seminal plasma
Acknowledgments This project was funded, in part, by a grant from the National Association of Animal Breeders. Dr. S. C. Barr provided valuable advice on aggregommetry. Eastern Artificial Insemination Cooperative provided the bull semen.
Theriogenology 46:649-658, 0 1996 by Elsewer Science
1996 Inc.
0093-691x/96/$15.00 PII SOO93-691X(96)00216-6
Theriogenology
650 INTRODUCTION
Several critical functions of spermatozoa such as the acrosome reaction and fertilization depend on membrane fusion events. These events require membrane fluidity. Spermatozoa are afforded this membrane fluidity by the high concentration in their plasmalemma of polyunsaturated fatty acids. The high concentration of unsaturated lipids renders spermatozoa exquisitely sensitive to damage by lipid peroxidation (1,16). One source of reactive oxygen species (superoxide anion, 02-; hydrogen peroxide, H202; hydroxyl radical, OH) is neutrophils recruited to the female tract in response to insemination. This response has been found to be rapid (occurring in less than 15 minutes) and exuberant, and occurs in all species examined to date (8,15,19). Oxidative damage not only has the potential to disrupt membrane fluidity and spermatozoa1 motility but may also destabilize DNA, with potential detrimental effects extending even beyond fertilization (5). A protective effect of seminal plasma against oxidative damage mediated by generation of reactive oxygen species by activated neutrophils would confer a functional advantage on spermatozoa. Such an effect has been reported for seminal plasma of humans and horses (14,2 1,22). Spermatozoa express unique differentiation antigens capable of eliciting immune responses in male and female animals (3). Antispermatozoa antibodies have been implicated as a cause of female infertility. It has been widely recognized that seminal plasma of several species exerts an immunosuppressive effect by inhibiting a range of lymphocyte functions, including the ability of T and B cells to proliferate in response to mitogen or antigen challenge and the ability of NK and cytotoxic T cells to recognize and destroy large T cells (3,23). Less attention has been paid to phagocytic and antigen-presenting cells, although it is known that human seminal plasma inhibits phagocytosis by neutrophils and macrophages and impairs the ability of these cells to recognize target antigens by means of surface cytophilic antibodies (3,22). We report here on experiments designed to examine the effect of seminal plasma from bulls on superoxide anion production by bovine neutrophils, and on homotypic aggregation of bovine neutrophils. Seminal plasma was also examined for a potential cytotoxic effect on bovine neutrophils. Lysis of oocytes, erythrocytes and mixed leukocyte suspensions by bovine seminal plasma has been reported (12,18). MATERIALS
AND METHODS
Ejaculates of normal semen were obtained by use of an artificial vagina from 6 bulls in good health and of established fertility. The bulls were housed in a commercial artificial insemination center. Within 90 min of collection, the semen samples were centrifuged at 3,000 x g for 30 min, and the seminal plasma was separated from the spermatozoa. Fresh seminal plasma was used for Experiment 1. The remaining seminal plasma was then stored at -70 “C for Experiments 2 and 3. Neutrophils were isolated from jugular venous blood samples obtained from healthy, lactating Holstein cows according to a modification of the method of Carlson and Kineko (7).
Theriogenology
651
Briefly, blood was obtained using acid citrate dextrose as anticoagulant and centrifuged at 100 x g for 15 min. The plasma, huffy coat and top half of the erythrocyte column were gently aspirated and discarded. Remaining erythrocytes were removed by flash hypotonic lysis by addition of 20 ml of cold (5 “C), sterile water for 60 sec. Isotonicity was restored by addition of 10 ml of 3 x Hanks Buffered Salt solution (HBSS) without calcium or magnesium. The volume of the mixture was made up to 45 ml with 1 x HBSS and centrifuged at 200 x g for 10 min. The supernatant was discarded, and the washing procedure repeated twice more. Isolated neutrophils were counted and resuspended at 2 x lo6 neutrophils/ml in HBSS with divalent cations. This method routinely yielded cell preparations consisting of more than 90% neutrophils (confirmed by examination of Diff-Quick-stained cytocentrifuge preparations) of greater than 90% viability as determined by trypan blue exclusion. For Experiment 1, neutrophil superoxide anion production was determined by the superoxide dismutase-inhibitable reduction of cytochrome C at 38 “C in the presence of 0, 4, 8 and 16 % seminal plasma with and without addition of phorbol myristate acetate (PMA) to activate the neutrophil oxidative burst (4,lO). The reaction mixture consisted of 1O6 neutrophils, 0.8 mg/ml ferricytochrome C, 0 or 100 rig/ml PMA, in 2.5 ml total volume. The reaction was allowed to proceed at 38 “C for 20 min and was stopped by placing samples on ice for 5 min. After centrifugation the absorbance of the supematant was measured at 550 nm. Production of superoxide anion was calculated according to Beer’s Law, ascribing an extension coefficient of 21.1 r&I-l cm -’ to cytochrome C (17). All samples were tested in duplicate. Seminal plasma from each bull was tested with neutrophils from 2 (different) random cows. In Experiment 2, the effect of seminal plasma on homotypic neutrophil aggregation was determined. Neutrophils were obtained as before, and suspended at 106/m1 with seminal plasma at a concentration of 0.2, 0.75,3.0, 12.0 and 45.0 % in a mixture of HBSS and Tyrode’s solution. One milliliter of this reaction mixture was prewarmed and placed into the reaction chamber of a ChronoLog aggregometer under constant agitation with a magnetic stir bar. After equilibration, aggregation was induced by addition of PMA to yield a final concentration of 50 rig/ml. Aggregation was expressed as the change in chamber impedance (in ohms) in 10 min. In Experiment 3, lo6 neutrophils were suspended with a final concentration of seminal plasma of 0, 2.5, 5.0, 10.0, 20.0 or 40.0 % in HBSS with divalent cations and incubated at 38 C for 60 min. Aliquots from each sample were removed at 0, 10, 20, 30 and 60 min and stained with trypan blue (0.2 %) for 10 min. The cells were then examined microscopically, and the proportion of cells excluding the dye was expressed as a percentage after counting 200 neutrophils. Data from Experiment 1 were examined by two-way analysis of variance, with bull and seminal plasma concentration as the independent variables and superoxide anion production as the dependent variable. When the analysis of variance indicated that statistically significant differences did, in fact, exist, they were further explored and examined for statistical significance by use of Dunnett’s method for multiple comparisons. Data from Experiment 2 were analyzed in
652
Theriogenology
essentially the same way except that one-way analysis of variance was used initially because only 1 independent variable (seminal plasma concentration) was used. For the data from Experiment 3, two-way analysis of variance for repeated measures was used initially; the independent variables were seminal plasma concentration and time. Dose-dependency was examined by determining Pearson’s product moment correlation coefficient between seminal plasma concentration and the independent variable in Experiments 1 and 2, and by multiple linear regression (to determine effect of time and seminal plasma concentration) in Experiment 3. For all measures, differences were considered statistically significant at P < 0.05. Calculations were performed on a microcomputer using commercially available software (Sigma&t, Jandel Scientific, Inc.). RESULTS Seminal plasma significantly diminished measured superoxide anion production by stimulated bovine neutrophils (P = 0.008; Figure 1). Stimulated superoxide anion production decreased from 14.73 + 0.29 (mean + pooled SEM) nmol / lo6 cells / 20 min in the absence of seminal plasma to 10.67 f 0.29, 9.41 f 0.29, and 7.17 + 0.29 nmol / lo6 cells / 20 min in the presence of 4, 8 or 16 % seminal plasma, respectively. Dose-dependency of this response was indicated by the correlation between seminal plasma concentration and superoxide anion production (r = -0.48; P = 0.0006). Dunnett’s test indicated that seminal plasma significantly depressed stimulated superoxide anion at 8 and 16 % (P < 0.05) relative to superoxide anion production in the absence of seminal plasma. There was no difference in unstimulated superoxide anion production by the neutrophils in the presence or absence of seminal plasma, indicating that seminal plasma itself did not stimulate the respiratory burst of neutrophils. The effect of seminal plasma donor was not significant (P = 0.61).
653
Theriogenology
0
4 sefhnal
Figure 1.
8 Flasma
16 (%i
The effect of seminalplasmaon the production of superoxideanion by bovine neutrophilsstimulatedby PMA. (Error barsrepresentstandarddeviation; n=6; P = 0.008.)
In Experiment2 seminalplasmadramaticallyreducedhomotypic neutrophil aggregation asmeasuredby the changein aggregometerchamberimpedanceover 10 min after applicationof the PMA stimulus(P < 0.0001; Figure 2). A concentrationof 0.2 % seminalplasmaeffectively abolishedneutrophilaggregation.This and all higher concentrationswere significantly different from 0 % seminalplasma(Dunnett’s test; P < 0.05). This effect was dose-dependent, with increasingseminalplasmaconcentrationsproducingprogressivelygreaternegative changesin aggregometerchamberimpedance.(Pearson’sproduct momentcorrelationcoefficient r = -0.53; P < 0.001). At higherseminalplasmaconcentrationschamberimpedancedecreasedrather than increasingor staying constant. One explanationfor this observationwould be disruption of neutrophil plasmalemmawith leakage of electrolytes into the extracellular medium, which promptedExperiment3.
654
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4
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3 2
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1
5 iti 5
0
z n. .-E k
-1
-2 -3
f
-4
E .E
-5
Et
s 6
Figure2.
0 (Control)
Seminal
0.2%
Plasma
0.75%
2.94%
11.75%
45%
Concentration
The effect of seminalplasmaon homotypic aggregationof bovine neutrophils after stimulationby PMA. Aggregation is measuredas the changein chamber impedancein ohmsover 10 minutesafter applicationof the stimulus. Data are presentedasthe meanand standarddeviation. (n = 6; P < 0.0000001.)
In Experiment 3 seminalplasmawas found to exert a significant cytotoxic effect on neutrophils,as determinedby their ability to exclude the vital dye trypan blue (Figure 3). The effect wassignificantly dependenton time (P < 0.0001)andconcentrationof seminalplasma(P < 0.0001)with a significant interactionbetweenthesevariables(P < O.OOOl),indicating that the effect of seminalplasmaconcentrationwasdependenton the durationof exposure. All seminal plasmaconcentrationsof 2.5 % or greaterhad a significantly (P < 0.05) detrimentaleffect on neutrophil viability after 20 min (40 % seminalplasma)or 30 min (20, 10, 5, 2.5 % seminal plasma). In neutrophilsnot exposedto seminalplasma,cell dye exclusionremainedabove 95 % for the durationof this experiment,and the influenceof time wasnot significant. Multiple linear regressionconfirmedthat the cytotoxic effect of seminalplasmaon bovine neutrophilsincreased with duration of exposure(P < 0.0001) and increasingseminalplasmaconcentration (P < 0.0001).
Theriogenology
655
120
80
60
40
20
0 0
Seminal
3
Plasma
Concentration
The effect of seminal plasma vital dye, trypan blue. Within each represent times 0, 10, 20, 30 and 60 standard error of the mean. (Effects (P < 0.0001) and time-by-seminal statistically significant.)
(%)
on the ability of bovine neutrophils to exclude the cluster of bars (seminal plasma concentration), bars minutes of incubation. Error bars represent pooled of seminal plasma concentration (P < O.OOOl), time plasma concentration interaction (P < 0.0001) are
DISCUSSION The experiments reported here collectively indicate a profound impairment of neutrophil function by bovine seminal plasma. The diminished cytochrome-c reduction observed in Experiment 1 could be due to free radical scavengers in the seminal plasma, or to impairment of the neutrophil capacity for oxidative metabolism. The presence of antioxidants in seminal plasma, including superoxide dismutase (14), and glutathione peroxidase (13) has been reported. Seminal plasma contains high concentrations of prostaglandins, and prostaglandin E2, in particular, has been found to suppress the oxidative burst of neutrophils and monocytes (28). Other workers have ascribed immunosuppressive activity to unidentified high or low molecular weight substances in seminal plasma (18,20,25,26,29).
656
Theriogenology
Homotypic neutrophil aggregation has been found to depend on expression of the Mac-l glycoprotein (CD1 lb/CD1 8) by neutrophils (6,24). This molecule also serves as the receptor for complement fragment C3bi, known as complement receptor 3 or CR3 (27). Function of this receptor mediates phagocytosis of organisms opsonized by complement. Impairment of aggregation is therefore an indirect indication of reduced or faulty expression of this molecule, and may indicate impaired phagocytic ability of the neutrophils exposed to seminal plasma. Negative changes in chamber impedance prompted speculation that seminal plasma may disturb neutrophil membrane integrity, allowing leakage of electrolytes from the cell. Impairment of cellular aggregation was confirmed to occur in addition to this possibility by microscopic examination of neutrophil suspensions stimulated with PMA in the presence or absence of seminal plasma (results not shown). Experiment 3 was designed to determine whether seminal plasma had a direct effect on neutrophil integrity. Exposure to seminal plasma reduced the ability of neutrophils to exclude the vital dye trypan blue. Results reported here may underestimate damage to neutrophils because, it was our impression that some cells were lysed completely, and were therefore not included in the determination of the percentage of cells able to exclude trypan blue. It has previously been reported that bovine seminal plasma has the ability to lyse erythrocytes and mixed suspensions of leukocytes (18) and oocytes (12). The cumulative results of this experiment indicate a profound impairment by seminal plasma of important indices of neutrophil function. The physiological advantage conferred by this mechanism remains largely speculative, and, although seminal plasma components adhere to sperm, it is not known what concentrations of seminal plasma are achieved in the bovine uterus after natural or artificial insemination. However, it seems very likely that the detrimental effect on neutrophils could well be an important factor in the transmission of venereal infections to female animals as well as the pathogenesis of infectious conditions of the male reproductive tract. Oxidative processes have the potential to damage the spermatozoa membranes by lipid peroxidation (2,11,14,16) but, perhaps more importantly, could also alter spermatozoa DNA (5). Protection of spermatozoa from oxidative damage may be an important function of seminal plasma. Residual amounts of seminal plasma may also serve to protect spermatozoa from premature phagocytosis by antigen presenting cells, which, in turn, may help prevent a humoral response to spermatozoa in the female tract. Although lipid peroxidation may injure spermatozoa cells, suggestions have recently been made that spermatozoa capacitation, hyperactivation and the acrosome reaction may be mediated by free oxygen radicals (2,9). Under this hypothesis, seminal plasma may be important both for protecting spermatozoa from exogenous sources of damaging free radicals, and preventing premature onset of pre-fertilization reactions such as capacitation and the acrosome reaction. The source and identity of the factors in seminal plasma responsible for impairment of neutrophil function are not yet known with certainty, although several suggestions have been made. If, as seems likely, accessory sex glands are responsible for production of these factors,
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Theriogenology
their presence may be important in the pathogenesis and persistence of infections of these organs. In this regard, infections of the vesicular glands of mature bulls are notoriously persistent and resistant to antimicrobial therapy. REFERENCES 1. Aitken RJ, Buckingham D, Harkiss D. Use of a xanthine oxidase free radical generating system to investigate the cytotoxic effects of reactive oxygen species on human spermatozoa. J Reprod Fertil 1993;97:441-50. 2. Aitken RJ, Clarkson JS, Fischel S. Generation of reactive oxygen species, lipid peroxidation, and human spermatozoa function. Biol Reprod 1989;40: 183-97. 3. Alexander NJ, Anderson DJ. Immunology of semen. Fertil Steril 1987;47:192-205. 4. Babior BM, Kipnes RS, Cumette JT. The production by leukocytes of superoxide, a potential bactericidal agent. J Clin Invest 1973;52:741-4. 5. Bernstein C. Sex as a response to oxidative DNA damage. In: Halliwell B, Arouma 01, (eds) DNA and free radicals. Chichester: Ellis Horwood, 1993; 193-2 10. 6. Bochsler PN, DorC M, Neilsen NR, Slauson DO. A monoclonal antibody-defined adhesion-related antigen on bovine neutrophils is required for neutrophil aggregation. Inflammation 1990;14:499-508. 7. Carlson GP, Kaneko JJ. Isolation of leukocytes from bovine peripheral blood. Proc Sot Exp Biol Med 1973;142:853-6. 8. Clark RA, Klebanoff SJ. Generation of a neutrophil chemotactic agent by spermatozoa: role of complement and regulation by seminal plasma factors. J Immunol 1976; 117: 1378-86. 9. De Lamirande E, Gagnon C. Human spermatozoa hyperactivation and capacitation as parts of an oxidative process. Free Radic Biol Med 1993; 14: 157-66. 10. DorC M, Slauson DO, Neilsen NR. Decreased respiratory burst activity in neonatal bovine neutrophils stimulated by protein kinase C agonists. Am J Vet Res 1991;52:375-SO. 11. Gagnon C, Iwasaki A, De Lamirande E, Kovalski N. Reactive oxygen species and human spermatozoa. Ann NY Acad Sci 1991;637:436-44. 12. Ijaz A, Hunter AG, Ayoub M. Lysis of oocytes by bovine spermatozoa and seminal plasma. J Dairy Sci 1989;72:3273-9. 13. Kantola M, Saaranen M, Vanha-Perttula T. Selenium and glutathione peroxidase in seminal plasma of men and bulls. J Reprod Fertil 1988$X3:785-94. 14. Kobayashi T, Miyazaki T, Natori M, Nozawa S. Protective role of superoxide dismutase in human spermatozoa motility: superoxide dismutase activity and lipid peroxide in human seminal plasma and spermatozoa. Hum Reprod 1991;66:987-91. 15. Kotilainen T, Huhtinen M, Katila T. Sperm-induced leukocytosis in the equine uterus. Theriogenology 1994;41:629-36. 16. Kovalski NN, De Lamirande E, Gagnon C. Reactive oxygen species generated by human neutrophils inhibit spermatozoa motility: protective effect of seminal plasma and scavengers. Fertil Steril 1992;58:809-16. 17. Massey V. The microestimation of succinate and the extinction coefficient of cytochrome-c. Biochem Biophys Acta 1959;34:255-8. 18. Matousek J, Stanek R. Immunosuppressive and leucolytic effect of phospholipid binding protein in bull seminal plasma. Anim Reprod Sci 1993;3 1: l-6.
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19. Pandya IJ, Cohen J. The leukocytic reaction of the human uterine cervix to spermatozoa. Fertil Stcril 1985;43 :4 17-2 1. 20. Perry ACF, Jones R, Hall L. Studies on a novel, secreted form of superoxide dismutase in rat epididymis. J Reprod Fertil, Abstr Series No. 10, 1993:44-Abstr. 84. 2 1. Scheytt AF, Gilbert RO. Stallion seminal plasma suppresses the oxidative burst of equine neutrophils. Proc Conf Res Workers Anim Dis 1993;74:8 abstr. 22. Schopf RI?, Schramm P, Benes P, Morsches B. Seminal plasma-induced suppression of the respiratory burst of polymorphonuclear leukocytes and monocytes. Andrologia 1984;16:124-8. 23. Skibinski G, Kelly RW, Harkiss D, James K. Immunosuppression by human seminal plasma - extracellular organelles (prostasomes) modulate activity of phagocytic cells. Am J Reprod Immunol 1992;28:97-103. 24. Slauson DO, Skrabalak DS, Neilsen NR, Zwahlen RD. Complement-induced equine neutrophil adhesiveness and aggregation. Vet Path01 1987;24:239-49. 25. Vallely PJ, Sharrard RM, Rees RC. The identification of factors in seminal plasma responsible for suppression of natural killer cell activity. Immunology 1988;63 :45 1-6. 26. Voutto ML, Peluso G, Mancino D, Colomra G, Facchiano A, Ielpo MT, Ravagnan G, Metafora S. Inhibition of interleukin-1 release and activity by the rat seminal vesicle protein SV-IV. J Leukoc Biol 1993;53:214-22. 27. Walport MJ. Complement. In: Roitt IM, Brostoff J, Male DK (eds), Immunology. London: Mosby, 1993;12.1-12.17. 28. Weitzman JB, Wells CE, Wright AH, Clark PA, Law SKA. The gene organisation of the human 2 integrin subunit (CD18). FEBS Lett 1991;294:97-103. 29. Wempe F, Wagner S, V.d.Kammer H, Krauhs E, Scheit KH. Seminalplasmin, the major basic protein of bull seminal plasma, is a secretory protein of the seminal vesicles. Biochim Biophys Acta Gen Subj 1990;1034:260-2.