Clin. exp. Immunol. (1991) 86, 550-556
Extracellular organelles (prostasomes) are immunosuppressive components of human semen R. W. KELLY, P. HOLLAND, G. SKIBINSKI, C. HARRISON*, L. MCMILLAN*, T. HARGREAVE* & K. JAMES* Medical Research Council Reproductive Biology Unit, and *Edinburgh University Department of Surgery, Wilkie Laboratories, Edinburgh, Scotland
(Acceptedfor publication 12 June 1991)
SUMMARY
Numerous reports have ascribed immunosuppressive activity to human seminal plasma and there is growing agreement that much of this activity can be accounted for by the very high levels of E series prostaglandins present (up to 300 pM 19-hydroxy prostaglandin E). However not all suppressive activity is due to prostaglandin since several reports have appeared of high molecular weight active substances and we have found that stripped seminal plasma is still effective in inhibiting the mitogeninduced proliferation of lymphocytes. In this study such immunosuppressive activity has been separated by molecular size fractionation and the activity has been found to be particulate and corresponded to the previously reported prostasomes. These are trilaminar to multilaminar vesicles (150 nm diameter) which are secreted by the prostate. Pure preparations of prostasomes inhibited mitogen-induced lymphoproliferation in a dose-dependent manner with a concentration of prostasomes equivalent to 40% of that seen in seminal fluid giving 69% suppression of thymidine incorporation. The suppressive activity survived boiling and therefore was unlikely to be due to enzymatic action associated with these organelles. Interaction with the accessory cells, involved in full development of the lymphoproliferation induced by mitogen, was indicated and this possibility was supported by the demonstration of a direct effect of prostasomes on macrophage function using a mouse macrophage cell line. The prostasomes in semen may play a complementary role to the prostaglandins in neutralizing the immune defences of the female reproductive tract. This combination would allow the alloantigenic spermatozoa the best chance of achieving fertilization, but at the same time leave the recipient open to any infection present in the semen. Keywords prostaglandin E prostasomes seminal plasma AIDS HPV
INTRODUCTION Human seminal plasma contains powerful immunosuppressive agents (Stites & Erickson, 1975) thought to be present in order to prolong the life of the spermatozoa, and to prevent hypersensitization of the female to the alloantigenic proteins present on the surface of the spermatozoa and in the seminal plasma (James & Hargreave, 1984). This would accord with seminal plasma's primary function of sustaining the spermatozoa and maximizing the chances of fertilization, and would account for the relatively low incidence of antibodies directed against sperm in women (Bronson, Cooper & Rosenfeld, 1984). The presence in semen of potent agents capable of suppressing cells of the female's immune system may yet prove to be the Achilles heel of reproduction in the primate, since transmission of disease in semen is a growing problem. The problem for primates may be accentuated by their receptivity to coitus, and therefore to the Correspondence: R. W. Kelly, Medical Research Council Reproductive Biology Unit, 37 Chalmers Street, Edinburgh EH3 9EW, UK.
alloantigenic spermatozoa, through the ovarian cycle, at all seasons and to a limited extent through pregnancy. Their immunosuppressive cover in the ejaculate may have been forced to adapt and certainly the primate ejaculate contains uniquely high levels of prostaglandins with the 19-hydroxy prostaglandins only found in primate semen (Kelly et al., 1976). Recently there has been some agreement that a major contribution to the immunosuppressive effect of human seminal plasma is provided by these prostaglandins (Tarter, CunninghamRundles & Koide, 1986; Vallely, Sharard & Rees, 1988; Quayle et al., 1989) which are present in huge concentrations in human semen, with PGEI and PGE2 present around 80 yM and 19hydroxy PGEI and 19-hydroxy PGE2 both around 300 pM (Templeton, Cooper & Kelly, 1978). These are several orders of magnitude above the levels found in an inflammatory situation (Higgs & Salmon, 1979) which would generally be regarded as suppressive (Goodwin, Bankhurst & Messner, 1977). Activity has been ascribed to the 19-hydroxy PGE fraction (Tarter et al., 1986), to the PGE fraction (Valleley et al., 1988) or to both
550
Extracellular organelles fractions (Quayle et al., 1989). All these studies have used the inhibition of natural killer (NK) cell function as their measure of suppression and such tests show that there is no activity remaining in human seminal plasma stripped of lipid (Tarter et al., 1987; Quayle et al., 1989). However, human seminal fluid is a complex mixture and there is evidence of other immunosuppressive agents present in human semen; high molecular weight suppressor substances have been reported (Marcus, Hess & Freisheim, 1983; Lord, Sensabaugh & Stites, 1977; Lee & Ha, 1989), and stripped human seminal plasma retains suppressive activity in lymphoproliferation assays (Quayle et al., 1989). We have investigated this activity by separation of human seminal fluid by molecular size chromatography and differential centrifugation and in the light of these results have investigated the immunosuppressive properties of the microparticulate prostasomes from human seminal plasma. MATERIALS AND METHODS
Seminal fluid Human seminal fluid was obtained from donors by masturbation; the spermatozoa were removed by centrifugation (100 g for 20 min) and the samples were stored at -20'C for up to 4 weeks.
Molecular weight fractionation of whole seminalfluid Frozen pooled seminal plasma (1 ml) was diluted 1:1 with phosphate-buffered saline (PBS) and centrifuged at 12 000 g for 3 min (Biofuge). The supernatant was injected onto a high resolution 6% cross-linked agarose column (Superose 6, HR 10/ 30, Pharmacia, Uppsala, Sweden) and eluted with PBS (0 05 M phosphate, 0-15 M NaCI) at 0 3 ml/min. Fractions of 0-6 ml were collected and eluate was monitored at 280 nm. Molecular weight markers used were; 'A', blue dextran (2 x 106 D); 'B', thyroglobulin (669 kD); 'C', catalase (230 kD), and 'D', RNAase (13-7 kD). Tritiated PGE2 (Amersham International, Amersham, UK) was added to the sample and was detected in fractions by liquid scintillation chromatography of 50 pI aliquots.
Lymphoproliferation assays The lymphoproliferation assay has been described by Quayle et al. (1989). Essentially, a preparation of peripheral blood mononuclear cells from healthy donors was prepared and 105 cells were used in each well of a 96-well plate. Proliferation of T cells was induced with phytohaemagglutinin (PHA) (Sigma, Poole, UK). The concentration of mitogen used was 2-5 yg/ml. Cells were allowed to proliferate for 48 h before pulsing with 1 pCi/well of tritiated thymidine (Amersham, TRA310). Cells were harvested with a Skatron semi-automatic cell harvester and the dried, glass-fibre discs were counted in 2 ml scintillant.
Preparation of protein-rich fraction of seminal fluid and separation of prostasomes Pooled, frozen seminal plasma (180 ml) was centrifuged at 10 000 g and the precipitate was discarded. The supernatant was cooled to 4 C and 80 ml of ice-cold saturated ammonium sulphate was added and stirring was continued at 4 C for 30 min. Of this suspension, 160 ml was centrifuged at 20 000 g for 20 min and the supernatant was discarded. The precipitate was suspended in 40 ml of PBS and was dispersed in a Potter Elvejhem homogenizer. This suspension was centrifuged at
551
100 000 g for 45 min. The supernatant was removed and chromatographed as above and the pellet was washed by resuspension and further centrifuged at 100 000 g and chromatographed similarly. The final suspension (10 ml) had a protein content of 9 25 mg/ml (equivalent to 514 pg/ml of original seminal plasma). Preparation of prostasomes by differential centrifugation Seminal plasma (40 ml) was centrifuged for 15 min at 10 000 g at 4 C to remove any debris and amorphous material in the samples and the pellet was discarded. The fluid was then centrifuged at 100 000 g for 1 h and the pellet was washed by resuspension in PBS and recentrifugation. The pellet was then finally resuspended in PBS (10 ml) by repeated aspiration and expulsion from a plastic pipette. The protein concentration of the suspension was 1-65 mg/ml (equivalent to 412 pg per ml seminal plasma). Gentamycin (25 pg/ml) was added to the preparation to prevent bacterial growth (Fig. 3a and b) and gentamycin at an equivalent concentration (0 5 pg/ml final) was added to all other treatments in these experiments. Boiled prostasomes were prepared by heating prostasome preparations to 100IC for 2 min. No clumping of the particles was observed. In an alternative procedure (Stegmayr & Ronquist, 1982), semen (80 ml) was centrifuged at 10 000 g (pellet discarded) and then at 1 00O0 g, and the high speed pellet resuspended in 10 ml PBS and chromatographed on a 120 mm x 30 mm diameter column of Sephadex G200 using PBS as the mobile phase. Fractions of 10 ml were collected and the two fractions eluting at the exclusion front were pooled. This material was white with suspended particles and had a u.v. spectrum showing maximum absorption below 230 nm and with only a minor shoulder at 280 nm. Such a pattern is consistent with particulate material with a low concentration of soluble protein. These fractions were further centrifuged at 100 000 g and the pellet suspended in 4 ml PBS. The final protein concentration was 1-9 mg/ml which was equivalent to 95 pg/ml original seminal plasma.
Phagocytosis of latex particles P388D 1 cells (American Type Culture Collection) were cultured in the presence of lipopolysaccharide (40 pg/ml) for 3 days prior to use in experiments. The effects of prostasomes, prostaglandins and human seminal plasma on phagocytosis were tested with the stimulated cells and latex particles of 3 1 pm diameter (Sigma), using 2 x 105 cells suspended in 0-2 ml culture medium (RPMI with 10% pooled human serum). The particle to cell ratio was 500:1. The mixture was incubated at 37 C for 60 min. After incubation, the cells were washed twice with cold PBS, cytocentrifuged and Giemsa stained. Results were determined as the mean percentage of phagocytosing cells, where cells containing three or more latex particles were considered positive. Each experiment was done with four replicates. Statistical analysis
Significant differences were determined by analysis of variance using the Newman Keuls procedure to assign significant differences. Cell viability The viability of cells unstimulated with lectin was checked using ethidium bromide and acridine orange staining (Parks et al.,
552
R. W. Kelly et al.
1962) after incubation with and without prostasomes and PGE2. Viability in all cases was greater than 90%.
Electron microscopy A sample of prostasomes prepared by direct differential centrifugation was fixed in glutaraldehyde in cacodylate buffer. Washing with buffer was followed by osmium tetroxide staining, washing, uranyl acetate treatment and embedding in epoxy resin.
Size distribution Size distribution of prostasomes (prepared by differential centrifugation and chromatography) was determined in a
0
> 2*5
-
1-0 103 104 105 106 107
Molecular weight
ccw N 0
A B
C
fluorescence activated cell sorter (EPICS, Coulter, Hialea, FL) using scattered light. Latex beads (Sigma) were used as size markers. RESULTS The chromatography of whole seminal fluid shows that the major suppressive action is seen in the low molecular weight region but that there is significant activity in the eluate corresponding to very high molecular weight (Fig. 1). A more concentrated preparation of this active material could be prepared by ammonium sulphate precipitation and redissolution of the proteins in saline. The material that then sedimented at 1 00O0 g was chromatographed and this preparation showed that the majority of the protein had a greater mol.wt than 2 x 106 D and the immunosuppressive activity was largely in this fraction with a smaller amount in the low mol.wt fraction (Fig. 2a). These early fractions were turbid, suggesting particulate material, most likely to be the prostasomes which have been reported to be present in human semen (Stegmayr & Ronquist, 1982). In confirming experiments, the suppressive activity could also be derived by direct centrifugation (Fig. 3). The particulate fraction was characterized as prostasomes by the following procedures. Sephadex chromatography of the 100 000 g pellet gave a milky-white fraction at the excluded front of the eluate. These fractions had a u.v. spectrum showing maximum absorption below 230 nm with a minor shoulder at 280 nm confirming the relative paucity of soluble protein. The protein concentration of the final preparation was 19 mg/ml, the majority of which was assumed to be within or on the surfaces of the
E
D
C
0
II
00
80rN I|0~~~~~~ c
Q
N
60!-
|
|
|\ PGE2
0
0
0
CL CL
40
1-
(I,
AB
C
D
0
20 F
o:
80
||
80 60
LO
40
CL I
I
0
10
20
30
40
50
~
~
v20 -20 0
Fig. 1. Separation of whole seminal fluid on the basis of molecular weight. Standard markers of molecular weight used were as follows. 'A', blue dextran (2 x 106 D); 'B', Thyroglobulin (669 kD); 'C', catalase (230 kD); and 'D', RNAase (13-7 kD). The point at which tritiated PGE2 elutes is indicated.
40
20 0
Fraction number
A
60
10 20 30 40 50 Fraction number
0
-20
0
10 20 30 40 50 Fraction number
Fig. 2. Separation by molecular weight of the two fractions (particulate (a) and soluble (b)) which comprise the protein rich fraction. Conditions and molecular weight markers as in Fig. 1.
Extracellular organelles 5000 r
60000
4000
>50000
3000
-, 40 000
2000
,, 30000
u
.n
-
C 0
10000 o
nI
I
PS2 Control Boil
PS c
Boil 2 E2 10-6
CL
30 000 20 000
100000 I) 0
_
a
.
1% sp
PS
0-2
0-3
04
Seminal plasma
Table 1. Lack of suppression of cytotoxicity of NK cells by prostasomes at two target/effector (T/E) ratios
I
Control
Spermine
10-6
M
100 000
Dilution
80000
T/E ratio cytotoxicity T/E ratio cytotoxicity
Experiment 1 Prostasomes 32% Prostasomes 16% Prostasomes 8% Control
60000 40000
_zz_-
Experiment 2 Prostasomes 32%
20 000 I
0
0-1
Fig. 4. Effect of increasing concentrations of prostasomes on mitogeninduced lymphoproliferation. Prostasomes were prepared by differential centrifugation and chromatography and concentrations are presented as the ratio of the concentration in the original seminal plasma, assuming 100% recovery (relative concentration). A concentration equivalent to that seen in whole seminal plasma would be 1 0. The effect of 1 % whole seminal plasma (which also contains prostaglandin) is shown for comparison.
40 000f-
H-
6
Relative concentration
4.0
_
&20000-
1000 (r
553
I
IZZ
I
I
*zz
I
I
I ZA AI
,
E2l0-6 1%/sp ps :50 Control Fig. 3. (a) A comparison of fresh and boiled prostasomes. Prostasomes were diluted 50 times in these experiments which was equivalent to 12 5% per ml of the seminal plasma content (assuming 100% recovery). (b) Prostasome suppression compared to that of PGE2 (10-6 M) and spermine (10-6 M). Prostasome effects can be enhanced further by the addition of PGE2. Spermine is totally inactive in this system since no exogenous amine oxidase (e.g. from fetal calf serum) is present. + 10-6 M PGE2; 0, no added PGE. (c) Mitogen (2 5 pg PHA per ml, final) was added 1 h prior to the addition of treatments (0) in order to reduce the possibility of prostasomes inactivating the lectin. addition of lectin at the same time as the treatments. Protein weights added in the prostasome treatments were 2-4 (prostasomes 1); 6 6 (prostasomes 2) and 26-4 pg/well.
Prostasomes 16% Prostasomes8% Control
1:30 1:30 1:30 1:30
36 4 435 34 2 37-7
1:15 1:15 1:15 1:15
29 3 323 27-0 351
1:20 1:20 1:20 1:20
12 1 116 114 13 7
1:10 1:10 1:10 1:10
35 47
60 78
Prostasomes were prepared as described for Fig. 3 (method 2). Prostasome concentrations are presented as percent of concentration in the original seminal plasma, assuming 100% recovery.
U,
U,
prostasomes. The material recovered after chromatography was effective in suppressing lymphocyte proliferation in a dosedependent manner (Fig. 4). A concentration of prostasomes equivalent to 40% of that seen in seminal fluid gave a 69% suppression of thymidine incorporation, similar to that seen with 1 % seminal plasma. Particle size analysis in a fluorescenceactivated cell sorter showed a distribution where the majority of the particles were below 800 nm in diameter with a median size between 100 and 200 nm (the median is reported as 150 nm
(Ronquist & Brody, 1985). Although a tail of apparently larger particles was present in the run using prostasomes, a similar tail was seen with some latex beads and this might reflect aggregation. In addition, electron microscopy (using osmium tetroxide staining which will visualize lipid as well as protein) demonstrated the presence of many intact (and some fragmented) particles around 150 nm diameter (Fig. 6). From a total of six preparations of prostasomes prepared either by direct differential centrifugation or by centrifugation/ chromatography, there was a suppression of 59-9% + 5 9 (mean + s.e.m.) using a preparation equivalent to 32% of the original concentration. The combined volume of semen used to prepare these prostasomes was 560 ml obtained from over 250 ejaculates. The soluble fraction obtained by ammonium sulphate precipitation and redissolution had the majority of the activity
R. W. Kelly et al.
554
Table 2. The effect of prostasomes on phagocytosis of latex particles by P388D1 cells (mean % of the phagocytosing cells s.d., n = 4)
Treatment Prostasomes 32% Prostasomes 16% Prostasomes 8% Prostaglandin E210-6 M Prostaglandin E210-8 Prostaglandin E210 -9 Seminal plasma 1% Seminal plasma 0-1% Control
Experiment I 8-5 + 34* 12-5 +4-3*
22-0+5.1* 6 3+4.1* 153+3.1* 32-4+4-2 2 5+3-8* 5-1 +4-4* 37-8 + 2-8
Experiment 2
8-6+2.8* 110 + 3-4* 29-6+4-4 6-1 +2-2* 9-2+4-8* 23-4+ 3-7
3.4+2-4* 6 6 + 5-3* 28-6+ 3-1
* Samples differ from control P
Extracellular organelles (prostasomes) are immunosuppressive components of human semen R. W. KELLY, P. HOLLAND, G. SKIBINSKI, C. HARRISON*, L. MCMILLAN*, T. HARGREAVE* & K. JAMES* Medical Research Council Reproductive Biology Unit, and *Edinburgh University Department of Surgery, Wilkie Laboratories, Edinburgh, Scotland
(Acceptedfor publication 12 June 1991)
SUMMARY
Numerous reports have ascribed immunosuppressive activity to human seminal plasma and there is growing agreement that much of this activity can be accounted for by the very high levels of E series prostaglandins present (up to 300 pM 19-hydroxy prostaglandin E). However not all suppressive activity is due to prostaglandin since several reports have appeared of high molecular weight active substances and we have found that stripped seminal plasma is still effective in inhibiting the mitogeninduced proliferation of lymphocytes. In this study such immunosuppressive activity has been separated by molecular size fractionation and the activity has been found to be particulate and corresponded to the previously reported prostasomes. These are trilaminar to multilaminar vesicles (150 nm diameter) which are secreted by the prostate. Pure preparations of prostasomes inhibited mitogen-induced lymphoproliferation in a dose-dependent manner with a concentration of prostasomes equivalent to 40% of that seen in seminal fluid giving 69% suppression of thymidine incorporation. The suppressive activity survived boiling and therefore was unlikely to be due to enzymatic action associated with these organelles. Interaction with the accessory cells, involved in full development of the lymphoproliferation induced by mitogen, was indicated and this possibility was supported by the demonstration of a direct effect of prostasomes on macrophage function using a mouse macrophage cell line. The prostasomes in semen may play a complementary role to the prostaglandins in neutralizing the immune defences of the female reproductive tract. This combination would allow the alloantigenic spermatozoa the best chance of achieving fertilization, but at the same time leave the recipient open to any infection present in the semen. Keywords prostaglandin E prostasomes seminal plasma AIDS HPV
INTRODUCTION Human seminal plasma contains powerful immunosuppressive agents (Stites & Erickson, 1975) thought to be present in order to prolong the life of the spermatozoa, and to prevent hypersensitization of the female to the alloantigenic proteins present on the surface of the spermatozoa and in the seminal plasma (James & Hargreave, 1984). This would accord with seminal plasma's primary function of sustaining the spermatozoa and maximizing the chances of fertilization, and would account for the relatively low incidence of antibodies directed against sperm in women (Bronson, Cooper & Rosenfeld, 1984). The presence in semen of potent agents capable of suppressing cells of the female's immune system may yet prove to be the Achilles heel of reproduction in the primate, since transmission of disease in semen is a growing problem. The problem for primates may be accentuated by their receptivity to coitus, and therefore to the Correspondence: R. W. Kelly, Medical Research Council Reproductive Biology Unit, 37 Chalmers Street, Edinburgh EH3 9EW, UK.
alloantigenic spermatozoa, through the ovarian cycle, at all seasons and to a limited extent through pregnancy. Their immunosuppressive cover in the ejaculate may have been forced to adapt and certainly the primate ejaculate contains uniquely high levels of prostaglandins with the 19-hydroxy prostaglandins only found in primate semen (Kelly et al., 1976). Recently there has been some agreement that a major contribution to the immunosuppressive effect of human seminal plasma is provided by these prostaglandins (Tarter, CunninghamRundles & Koide, 1986; Vallely, Sharard & Rees, 1988; Quayle et al., 1989) which are present in huge concentrations in human semen, with PGEI and PGE2 present around 80 yM and 19hydroxy PGEI and 19-hydroxy PGE2 both around 300 pM (Templeton, Cooper & Kelly, 1978). These are several orders of magnitude above the levels found in an inflammatory situation (Higgs & Salmon, 1979) which would generally be regarded as suppressive (Goodwin, Bankhurst & Messner, 1977). Activity has been ascribed to the 19-hydroxy PGE fraction (Tarter et al., 1986), to the PGE fraction (Valleley et al., 1988) or to both
550
Extracellular organelles fractions (Quayle et al., 1989). All these studies have used the inhibition of natural killer (NK) cell function as their measure of suppression and such tests show that there is no activity remaining in human seminal plasma stripped of lipid (Tarter et al., 1987; Quayle et al., 1989). However, human seminal fluid is a complex mixture and there is evidence of other immunosuppressive agents present in human semen; high molecular weight suppressor substances have been reported (Marcus, Hess & Freisheim, 1983; Lord, Sensabaugh & Stites, 1977; Lee & Ha, 1989), and stripped human seminal plasma retains suppressive activity in lymphoproliferation assays (Quayle et al., 1989). We have investigated this activity by separation of human seminal fluid by molecular size chromatography and differential centrifugation and in the light of these results have investigated the immunosuppressive properties of the microparticulate prostasomes from human seminal plasma. MATERIALS AND METHODS
Seminal fluid Human seminal fluid was obtained from donors by masturbation; the spermatozoa were removed by centrifugation (100 g for 20 min) and the samples were stored at -20'C for up to 4 weeks.
Molecular weight fractionation of whole seminalfluid Frozen pooled seminal plasma (1 ml) was diluted 1:1 with phosphate-buffered saline (PBS) and centrifuged at 12 000 g for 3 min (Biofuge). The supernatant was injected onto a high resolution 6% cross-linked agarose column (Superose 6, HR 10/ 30, Pharmacia, Uppsala, Sweden) and eluted with PBS (0 05 M phosphate, 0-15 M NaCI) at 0 3 ml/min. Fractions of 0-6 ml were collected and eluate was monitored at 280 nm. Molecular weight markers used were; 'A', blue dextran (2 x 106 D); 'B', thyroglobulin (669 kD); 'C', catalase (230 kD), and 'D', RNAase (13-7 kD). Tritiated PGE2 (Amersham International, Amersham, UK) was added to the sample and was detected in fractions by liquid scintillation chromatography of 50 pI aliquots.
Lymphoproliferation assays The lymphoproliferation assay has been described by Quayle et al. (1989). Essentially, a preparation of peripheral blood mononuclear cells from healthy donors was prepared and 105 cells were used in each well of a 96-well plate. Proliferation of T cells was induced with phytohaemagglutinin (PHA) (Sigma, Poole, UK). The concentration of mitogen used was 2-5 yg/ml. Cells were allowed to proliferate for 48 h before pulsing with 1 pCi/well of tritiated thymidine (Amersham, TRA310). Cells were harvested with a Skatron semi-automatic cell harvester and the dried, glass-fibre discs were counted in 2 ml scintillant.
Preparation of protein-rich fraction of seminal fluid and separation of prostasomes Pooled, frozen seminal plasma (180 ml) was centrifuged at 10 000 g and the precipitate was discarded. The supernatant was cooled to 4 C and 80 ml of ice-cold saturated ammonium sulphate was added and stirring was continued at 4 C for 30 min. Of this suspension, 160 ml was centrifuged at 20 000 g for 20 min and the supernatant was discarded. The precipitate was suspended in 40 ml of PBS and was dispersed in a Potter Elvejhem homogenizer. This suspension was centrifuged at
551
100 000 g for 45 min. The supernatant was removed and chromatographed as above and the pellet was washed by resuspension and further centrifuged at 100 000 g and chromatographed similarly. The final suspension (10 ml) had a protein content of 9 25 mg/ml (equivalent to 514 pg/ml of original seminal plasma). Preparation of prostasomes by differential centrifugation Seminal plasma (40 ml) was centrifuged for 15 min at 10 000 g at 4 C to remove any debris and amorphous material in the samples and the pellet was discarded. The fluid was then centrifuged at 100 000 g for 1 h and the pellet was washed by resuspension in PBS and recentrifugation. The pellet was then finally resuspended in PBS (10 ml) by repeated aspiration and expulsion from a plastic pipette. The protein concentration of the suspension was 1-65 mg/ml (equivalent to 412 pg per ml seminal plasma). Gentamycin (25 pg/ml) was added to the preparation to prevent bacterial growth (Fig. 3a and b) and gentamycin at an equivalent concentration (0 5 pg/ml final) was added to all other treatments in these experiments. Boiled prostasomes were prepared by heating prostasome preparations to 100IC for 2 min. No clumping of the particles was observed. In an alternative procedure (Stegmayr & Ronquist, 1982), semen (80 ml) was centrifuged at 10 000 g (pellet discarded) and then at 1 00O0 g, and the high speed pellet resuspended in 10 ml PBS and chromatographed on a 120 mm x 30 mm diameter column of Sephadex G200 using PBS as the mobile phase. Fractions of 10 ml were collected and the two fractions eluting at the exclusion front were pooled. This material was white with suspended particles and had a u.v. spectrum showing maximum absorption below 230 nm and with only a minor shoulder at 280 nm. Such a pattern is consistent with particulate material with a low concentration of soluble protein. These fractions were further centrifuged at 100 000 g and the pellet suspended in 4 ml PBS. The final protein concentration was 1-9 mg/ml which was equivalent to 95 pg/ml original seminal plasma.
Phagocytosis of latex particles P388D 1 cells (American Type Culture Collection) were cultured in the presence of lipopolysaccharide (40 pg/ml) for 3 days prior to use in experiments. The effects of prostasomes, prostaglandins and human seminal plasma on phagocytosis were tested with the stimulated cells and latex particles of 3 1 pm diameter (Sigma), using 2 x 105 cells suspended in 0-2 ml culture medium (RPMI with 10% pooled human serum). The particle to cell ratio was 500:1. The mixture was incubated at 37 C for 60 min. After incubation, the cells were washed twice with cold PBS, cytocentrifuged and Giemsa stained. Results were determined as the mean percentage of phagocytosing cells, where cells containing three or more latex particles were considered positive. Each experiment was done with four replicates. Statistical analysis
Significant differences were determined by analysis of variance using the Newman Keuls procedure to assign significant differences. Cell viability The viability of cells unstimulated with lectin was checked using ethidium bromide and acridine orange staining (Parks et al.,
552
R. W. Kelly et al.
1962) after incubation with and without prostasomes and PGE2. Viability in all cases was greater than 90%.
Electron microscopy A sample of prostasomes prepared by direct differential centrifugation was fixed in glutaraldehyde in cacodylate buffer. Washing with buffer was followed by osmium tetroxide staining, washing, uranyl acetate treatment and embedding in epoxy resin.
Size distribution Size distribution of prostasomes (prepared by differential centrifugation and chromatography) was determined in a
0
> 2*5
-
1-0 103 104 105 106 107
Molecular weight
ccw N 0
A B
C
fluorescence activated cell sorter (EPICS, Coulter, Hialea, FL) using scattered light. Latex beads (Sigma) were used as size markers. RESULTS The chromatography of whole seminal fluid shows that the major suppressive action is seen in the low molecular weight region but that there is significant activity in the eluate corresponding to very high molecular weight (Fig. 1). A more concentrated preparation of this active material could be prepared by ammonium sulphate precipitation and redissolution of the proteins in saline. The material that then sedimented at 1 00O0 g was chromatographed and this preparation showed that the majority of the protein had a greater mol.wt than 2 x 106 D and the immunosuppressive activity was largely in this fraction with a smaller amount in the low mol.wt fraction (Fig. 2a). These early fractions were turbid, suggesting particulate material, most likely to be the prostasomes which have been reported to be present in human semen (Stegmayr & Ronquist, 1982). In confirming experiments, the suppressive activity could also be derived by direct centrifugation (Fig. 3). The particulate fraction was characterized as prostasomes by the following procedures. Sephadex chromatography of the 100 000 g pellet gave a milky-white fraction at the excluded front of the eluate. These fractions had a u.v. spectrum showing maximum absorption below 230 nm with a minor shoulder at 280 nm confirming the relative paucity of soluble protein. The protein concentration of the final preparation was 19 mg/ml, the majority of which was assumed to be within or on the surfaces of the
E
D
C
0
II
00
80rN I|0~~~~~~ c
Q
N
60!-
|
|
|\ PGE2
0
0
0
CL CL
40
1-
(I,
AB
C
D
0
20 F
o:
80
||
80 60
LO
40
CL I
I
0
10
20
30
40
50
~
~
v20 -20 0
Fig. 1. Separation of whole seminal fluid on the basis of molecular weight. Standard markers of molecular weight used were as follows. 'A', blue dextran (2 x 106 D); 'B', Thyroglobulin (669 kD); 'C', catalase (230 kD); and 'D', RNAase (13-7 kD). The point at which tritiated PGE2 elutes is indicated.
40
20 0
Fraction number
A
60
10 20 30 40 50 Fraction number
0
-20
0
10 20 30 40 50 Fraction number
Fig. 2. Separation by molecular weight of the two fractions (particulate (a) and soluble (b)) which comprise the protein rich fraction. Conditions and molecular weight markers as in Fig. 1.
Extracellular organelles 5000 r
60000
4000
>50000
3000
-, 40 000
2000
,, 30000
u
.n
-
C 0
10000 o
nI
I
PS2 Control Boil
PS c
Boil 2 E2 10-6
CL
30 000 20 000
100000 I) 0
_
a
.
1% sp
PS
0-2
0-3
04
Seminal plasma
Table 1. Lack of suppression of cytotoxicity of NK cells by prostasomes at two target/effector (T/E) ratios
I
Control
Spermine
10-6
M
100 000
Dilution
80000
T/E ratio cytotoxicity T/E ratio cytotoxicity
Experiment 1 Prostasomes 32% Prostasomes 16% Prostasomes 8% Control
60000 40000
_zz_-
Experiment 2 Prostasomes 32%
20 000 I
0
0-1
Fig. 4. Effect of increasing concentrations of prostasomes on mitogeninduced lymphoproliferation. Prostasomes were prepared by differential centrifugation and chromatography and concentrations are presented as the ratio of the concentration in the original seminal plasma, assuming 100% recovery (relative concentration). A concentration equivalent to that seen in whole seminal plasma would be 1 0. The effect of 1 % whole seminal plasma (which also contains prostaglandin) is shown for comparison.
40 000f-
H-
6
Relative concentration
4.0
_
&20000-
1000 (r
553
I
IZZ
I
I
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E2l0-6 1%/sp ps :50 Control Fig. 3. (a) A comparison of fresh and boiled prostasomes. Prostasomes were diluted 50 times in these experiments which was equivalent to 12 5% per ml of the seminal plasma content (assuming 100% recovery). (b) Prostasome suppression compared to that of PGE2 (10-6 M) and spermine (10-6 M). Prostasome effects can be enhanced further by the addition of PGE2. Spermine is totally inactive in this system since no exogenous amine oxidase (e.g. from fetal calf serum) is present. + 10-6 M PGE2; 0, no added PGE. (c) Mitogen (2 5 pg PHA per ml, final) was added 1 h prior to the addition of treatments (0) in order to reduce the possibility of prostasomes inactivating the lectin. addition of lectin at the same time as the treatments. Protein weights added in the prostasome treatments were 2-4 (prostasomes 1); 6 6 (prostasomes 2) and 26-4 pg/well.
Prostasomes 16% Prostasomes8% Control
1:30 1:30 1:30 1:30
36 4 435 34 2 37-7
1:15 1:15 1:15 1:15
29 3 323 27-0 351
1:20 1:20 1:20 1:20
12 1 116 114 13 7
1:10 1:10 1:10 1:10
35 47
60 78
Prostasomes were prepared as described for Fig. 3 (method 2). Prostasome concentrations are presented as percent of concentration in the original seminal plasma, assuming 100% recovery.
U,
U,
prostasomes. The material recovered after chromatography was effective in suppressing lymphocyte proliferation in a dosedependent manner (Fig. 4). A concentration of prostasomes equivalent to 40% of that seen in seminal fluid gave a 69% suppression of thymidine incorporation, similar to that seen with 1 % seminal plasma. Particle size analysis in a fluorescenceactivated cell sorter showed a distribution where the majority of the particles were below 800 nm in diameter with a median size between 100 and 200 nm (the median is reported as 150 nm
(Ronquist & Brody, 1985). Although a tail of apparently larger particles was present in the run using prostasomes, a similar tail was seen with some latex beads and this might reflect aggregation. In addition, electron microscopy (using osmium tetroxide staining which will visualize lipid as well as protein) demonstrated the presence of many intact (and some fragmented) particles around 150 nm diameter (Fig. 6). From a total of six preparations of prostasomes prepared either by direct differential centrifugation or by centrifugation/ chromatography, there was a suppression of 59-9% + 5 9 (mean + s.e.m.) using a preparation equivalent to 32% of the original concentration. The combined volume of semen used to prepare these prostasomes was 560 ml obtained from over 250 ejaculates. The soluble fraction obtained by ammonium sulphate precipitation and redissolution had the majority of the activity
R. W. Kelly et al.
554
Table 2. The effect of prostasomes on phagocytosis of latex particles by P388D1 cells (mean % of the phagocytosing cells s.d., n = 4)
Treatment Prostasomes 32% Prostasomes 16% Prostasomes 8% Prostaglandin E210-6 M Prostaglandin E210-8 Prostaglandin E210 -9 Seminal plasma 1% Seminal plasma 0-1% Control
Experiment I 8-5 + 34* 12-5 +4-3*
22-0+5.1* 6 3+4.1* 153+3.1* 32-4+4-2 2 5+3-8* 5-1 +4-4* 37-8 + 2-8
Experiment 2
8-6+2.8* 110 + 3-4* 29-6+4-4 6-1 +2-2* 9-2+4-8* 23-4+ 3-7
3.4+2-4* 6 6 + 5-3* 28-6+ 3-1
* Samples differ from control P
