JournalofReproductive Immunology, 18 (1990) 123--137
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Elsevier Scientific Publishers Ireland Ltd.
JRI 00674
G D A - J / F 7 monoclonal antibody: A new marker for sperm cell precursors in human semen A. J a s s i m Department of Immunology, London Hospital Medical College, Turner Street, London E1 2AD (U.K.) (Accepted for publication 16 March 1990)
Summary Germ cells isolated from semen of oligospermic human donors were found to react with GDA-J/F7 monoclonal antibody (MoAb). Their reactions with this antibody were demonstrated by using fluorescein activated cell sorter (FACS) analysis and indirect immunofluorescence (IIF) test. In the IIF test, the MoAb recognised an antigen on the surface of the sperm cell precursors (SpP) as well as on mature spermatozoa. The specificity of the antibody reaction with the SpP was further confirmed by immunoelectron microscopy. The MoAb did not react with peripheral blood lymphocytes or polymorphonuclear cells but did show cross-reactivity with monocytes. This antibody therefore provides the first marker for the SpP and could be used as a probe for their distinction from leucocytes. This could have clinical application in seminal analysis. Key words: sperm cell precursors; surface antigen; monoclonal antibody;
human semen.
Introduction The development o f monoclonal antibody technology has greatly helped in the characterisation and identification o f a large number o f cellular antigens. Some o f these antigens have been found to be specific for a particular tissue or a group o f cells; consequently the antibodies can be used as markers 0165-0378/90/$03.50 © 1990Elsevier Scientific Publishers Ireland Ltd. Pubhshed and Printed in Ireland
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for their identification, or even the classification of one group of cells into various subsets. In the field of reproduction, monoclonal antibodies (MoAbs) have been extensively used to study various biological phenomena, including germ cell differentiation (Jassim and Festenstein, 1987a), fertilisation and sperm-egg interactions (Mandelbaum et al., 1986; Salonen and Kallajoki, 1987; Moore et al., 1987), detection of abnormal germ cells in human semen (Jassim and Festenstein, 1988; Jassim et al., 1990) and others (Bellve and Moss, 1983; Yan et al., 1987). With few exceptions, most studies on human germ cells have concentrated on the use of MoAbs to analyse antigens on mature sperm (see Jassim and Festenstein, 1987a). Thus, immunological markers for SpP have been lacking. Consequently, in previous studies on the cellular analysis of human semen (EI-Demiry et al., 1986a,b; Wolff and Anderson, 1988; Witkin and Goldstein, 1988) the MoAbs used were only those against leucocytes and their various subsets, i.e. no marker for the germ cell was used. In this report a MoAb for the identification of the SpP is described, together with its potential clinical application in seminal analysis as a probe for the distinction between these cells and the leucocytes. Materials and Methods
Monoclonai antibodies (MoAbs) The MoAbs used in this study are listed in Table 1. All the antibodies which were used as supernatants were of mouse origin. The MoAbs against leucocytes and their different subsets (Table 1), as shown previously (Jassim et al., 1989a) and in this study (see Results), show no cross reaction with germ cells or subcellular particles (cf. Wolff and Anderson, 1988).
TABLE I MoAbs used in the characterisation of NCOS.
MoAb
Reference
LP34 G A P 8.3 GRMI 63D3 OKT3 W6/32 L243 GDA-J,.F7
(I. Leigh) Berger et al., 1981 Ruiz-Cabello et al., 1987 Ugolini et al., 1980 H o f f m a n et al., 1980 Parham et al., 1979 Lampson and L e w , 1980 Jassim and Festenstein 1987a
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Sperm donors The oligospermic donors used in this study were medical students who were previously shown to produce large numbers of nucleated cells other than sperm fNCOS) and low counts of sperm in their semen (Jassim and Festenstein, 1987b). In addition, 7 normospermic donors whose semen contained leucocytes (as characterised herein) were also included.
Separation of NCOS and peripheral blood leucocytes (PBL) The method for the separation of the NCOS from human semen and PBL from peripheral blood has been described previously (Jassim and Festenstein, 1987b). Basically, the technique involves the use of ficoll/triosil as the density gradient. The rate of NCOS recovery by using this method exceeds 90070 and the contamination with sperm is less than 10070. This has been demonstrated previously (Jassim and Festenstein, 1987b; Jassim et al., 1989a) as well as in the present study (see Figs. 1 and 2). The use of viable cells reduces the cross reaction of MoAbs with unrelated cytoplasmic antigens inside the germ cells (Jassim et al., 1989b).
Indirect immunofluorescence (IIF) test and FA CS analysis Two million washed cells (NCOS, PBL or sperm) were incubated with 100 /al of MoAb for 1 h at 37 °C. The cells were washed twice with phosphate buffered saline (PBS) and then incubated with 4/A of rabbit F(ab) 2 antimouse immunoglobulin conjugated to fluorescein isothiocyanate (Sigma) for 30 rain at 20 °C. The samples were finally washed 3 times with PBS and examined by using a fluorescence microscope a n d / o r subjected to FACS analysis. Flow cytometry was performed using a Coulter Epics C to measure excitation at 488 nm on a 10,000 cell count. Lymphocyte, monocyte and granulocyte cell populations were gated separately. Background fluorescence was set in each experiment by assaying cells incubated with the fluorescein-conjugated antibody only, i.e. without the primary MoAb. For testing LP34 MoAb, which recognises cytokeratin intermediate filaments, the cells were first fixed with 50:50 methanol/acetone before the addition of the antibody.
Immunoelectron microscopy Four million washed NCOS obtained from an oligospermic donor were incubated with 200/al of GDA-J/F7 MoAb for 1 h at 37 °C. The cells were washed twice with PBS and then incubated with 10 ~I of colloidal gold goat anti-mouse immunoglobulin (size 5 nm, Janssen) at 37°C for 50 min with frequent agitation. The NCOS were finally washed 3 times with PBS and fixed with 2°70 gluteraldehyde in PBS. Further processing of the samples for
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O Fig. I. Indirect immunofluorescence test of NCOS from an oligospermic donor ~ith GDA-J F7 MoAb: a, phase contrast; b, fluorescence microscopy. Note the surface labelling of the cells v, ith the antibod.~ and variation in the pattern of the immunofluorescence staining, dotted (curxed arrow); circular 0ong arrow); polar (arrow head) or interrupted circle (small arrowl. ( × 210).
Fig. 2. Indirect immunofluorescence test of NCOS from an oligospermic donor with W6/32 (a,b) and G A P 8.3 (c,d). The two antibodies failed to react with germ cells (cf. Fig. I) but stained occasional cells (arrows) indicating their origin from leucocytes. These cells showed the morphology of lymphocytes. (a,b) x 330; (c,d} x 210.
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electron microscopy was as described previously (Jassim and Festenstein, 1987b). Results
Reaction o f G D A - J / F 7 M o A b with germ cells in semen o f oligospermic donors Following their separation on a ficoll density gradient, the NCOS were tested with GDA-J/F7 MoAb using the IIF technique. The antibody produced strong immunofluorescence around the surface of 90--100°70 of the NCOS. The pattern of cell staining was variable, showing dotted, circular, polar or interrupted circle immunofluorescence (Fig. 1). This was noticed on cells which differed in size as well as internal structures. Using the IIF technique, the NCOS were also tested with a panel of tissuespecific MoAbs. In these experiments, the anti-leucocyte (and their subsets), anti-epithelial cells, anti-HLA Class I and anti-HLA-DR MoAbs failed to react with the NCOS. However, following the screening of a large number of NCOS, occasional cells were found positive with anti-leucocyte, anti-lymphocyte and anti-MHC Class I MoAbs. These cells showed the morphology of leucocytes and were probably lymphocytes (Fig. 2). Similar results were obtained by using FACS analysis where all the
L :L L .
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Fig. 3. FACS analysis of the NCOS from an oligospermic donor with tissue specific MoAbs: a, RPMI; b, LP34; c, GAP 8.3; d, GRMI; e, 63D3; f, OKT3; g, W6/32; h, L243; i, GDA-J/F7. The failure of the antiepithelial, anti-leucocytes and their subsets and anti-MHC Class I and HLA-DR MoAbs to react with the NCOS indicates their germinal origin. GDA-J/F7 MoAb reacted strongly with these cells (see also Table 2).
C
128 TABLE 2 FACS analysis of NCOS from oligospermic donors with tissue-specific MoAbs. The reactivity of the anti-epithelial, anti-leucocyte and their subsets and anti-MHC Class I and HLA-DR MoAbs with less than !% NCOS indicates their germ cell origin (see Fig. 3). MoAb
Specificity
% of positive cell
RPMI LP34 G A P 8.3 GRMI 63D3 OKT3 W6/32 L243 GDA-J/F7
negative control epithelial cells (keratin) leucocytes granulocytes and NK cells monocytes T lymphocytes Class I M H C antigens HLA-DR germ cells and monocytes
0.10 0.18 0.34 0.68 0.38 0.6 I 0.58 0.32 75.01
A
L~
L L L
B
a
I
i
b
C
dL
C
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d
Fig. 4. FACS analysis of the reactions of G D A - J / F 7 and anti-leucocyte M o A b s with NCOS (A) and PBL (B) of an oligospermic donor: a, G A P 8.3; b, GRMI; c, OKT3; d, 63D3; e, GDA-J/FT. Note the strong reaction of the NCOS but not the PBL with G D A - J / F 7 MoAb. The NCOS, unlike PBL, did not react with the anti-leucocyte (and their subsets) M o A b s (see also Table 3).
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MoAbs reacted with less than 1°70 of NCOS except GDA-J/F7. This antibody reacted with 75--100070 of NCOS. The results of one representative experiment using FACS analysis are shown in Table 2 and Fig. 3. In this particular experiment, the GDA-J/F7 MoAb reacted with 75% of the NCOS (Table 2). Figure 4 and Table 3 show a comparison of the reactivity of the MoAbs with the NCOS and PBL of the same donor. The results revealed the reaction of GDA-J/F7 with 78°70 of the NCOS while the anti-leucocyte MoAb (and antibodies against their various subsets) reacted with less than 1°70 of NCOS, i.e. similar to that of the negative control (data not shown). This was in contrast to their reactions with the PBL where the G A P 8.3 MoAb reacted with 99°70 of the cells while the GDA-J/F7 showed a reaction of 6°70 only (this, as discussed later, was against monocytes). The MoAbs against the leucocyte subpopulations reacted with the PBL in percentages corresponding to their specificities.
Reaction of GDA-J/F7 MoAb with leucocytes in semen o f normospermic donors The leucocytes in the semen of the normospermic donors were identified by using tissue specific MoAbs and anti-MHC Class I and HLA-DR antibodies as additional markers. The cells were tested by FACS analysis and IIF microscopy. Using the IIF technique, the anti-leucocyte and anti-MHC Class I MoAbs reacted with 82--95°70 of NCOS (Table 4). The GDA-J/F7 MoAb, on the other hand, showed a reaction with 7--130/0 of NCOS. In these donors, there was a tendency for the OKT3 antibody to react with higher percentages of NCOS than GRMI. TABLE3 FACS analysis of NCOS and PBL of an oligospermic donor with G D A - J / F 7 and anti-leucocyte MoAbs. Note the strong reaction o f the NCOS but not the PBL with G D A - J / F 7 . The NCOS, unlike the PBL, did not react with the anti-leucocyte (and their subsets) M o A b s (see Fig. 4). The negative control was less than ! % (data not shown). MOAb
NCOS
PBL
G A P 8.3' GRMI OKT3 63D3 GDA-J/F7
0.47 b 0.41 0.28 0.37 78.00
99.7 21.0 58.0 2.0 6.4
' For specificity of the MoAbs, see Table 2. b Numbers represent percentage of positive cells.
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TABLE 4 Characterisation of NCOS from 7 normosperic donors with MoAbs by |IF microscopy. The reaction of the anti-leucocyte and anti-MHC Class I MoAbs with the NCOS indicate that the majority of the cells were leucoc}ae in origin. G D A - J / F 7 showed minimal reactivity. MoAb
RPMP LP34 GAP 8.3 GRMI 63 D3 OKT3 W6,, 32 L243 GDA-J ,"F7
NCOS from donor A
B
C
D
E
F
G
It 3 90 17 25 50 95 25 8
0 2 85 20 23 44 90 20 7
I I 82 45 30 I0 87 30 13
2 2 95 18 28 40 91 30 I0
2 I 87 21 18 50 93 25 8
1 2 90 15 28 39 88 30 7
2 I 95 18 20 45 90 26 7
For specificity of the MoAbs, see Table 2. ° Numbers represent percentage of positive cells.
d
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g
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~
e
h
~_
_
r
Fig. 5. FACS analysis of the NCOS from a normospermic donor with tissue specific MoAbs: a, RPMI; b, LP34; c, GAP 8.3; d, GRMI; e, 63D3; f, OKT3; g, W6/32; h, L243; l, GDA-J/F7. The reactions of the anti-leucocytes and their subsets and anti-MHC Class I and HLA-DR MoAbs indicate that the majority of the NCOS were leucocytes in origin. G D A - J / F 7 MoAb showed Iov, reactivity (see also Table 5).
131 TABLE 5 FACS analysis o f NCOS from normospermic donors with tissue-specific MoAbs. The reactivity of the anti-leucocytes and their subsets and anti-MHC Class I and HLA-DR MoAbs with the NCOS indicates that the majority of the NCOS were leucocytes (see Fig. 5). MoAB
Specificity
% of positive cell
RPMI LP34 G A P 8.3 GRM I 63D3 OKT3 W6/32 L243 GDA-J/F7
negative control epithelialcells(keratin) leucocytes granulocytes and N K cells monocytes T lymphocytes Class I MHC antigens HLA-DR germ cells and monocytes
0,86 0.93 90.01 16.46 21.42 6.50 63.40 16.20 8. l0
The FACS analysis showed results comparable to those obtained by IIF microscopy. Thus, the anti-leucocyte and anti-Class I HLA MoAbs reacted with 60--95070 of the NCOS, while the antibodies against the various leucocyte subsets recognised cells in percentages corresponding to their specificities. The anti-epithelial LP34 MoAb reacted with less than 1o70 of NCOS. The GDA-J/F7 MoAb in all these experiments showed a reaction with 5-15o70 of NCOS. The results of one such experiment using FACS technique are shown in Fig. 5 and Table 5. In contrast to the reaction of the anti-leucocyte MoAb with 90°70 of NCOS, the GDA-J/F7 antibody reacted with only 8070 of the cells (Table 5).
Fig. 6. Indirect immunofluorescence test of sperm with GDA-J/F7 MoAb: a, phase contrast; b, fluores-
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Reaction o f G D A - J / F 7 M o A b with sperm Ejaculated sperm from normospermic and oligospermic donors were tested with GDA-J/F7 MoAb using the IIF test. The antibody produced patchy fluorescence along the entire surface of 90--100°/0 sperm of both groups of donors (Fig. 6). Reaction o f G D A - J / F 7 M o A b with dried germ cells Germ cells obtained from seminal samples of oligospermic donors were dried onto slides. These cells were tested with GDA-J/F7 MoAb using the IIF technique. This test was also used to stain frozen sections of normal adult testes. The results showed the failure of the antibody to react with the dried germ cells. Reaction o f G D A - J / F 7 M o A b with PBL By using the IIF technique, G D A - J / F 7 MoAb stained 3--10°70 of PBL. The cells showed dotted immunofluorescence around their surface. Similar results were obtained when the samples were tested using the FACS technique (Fig. 4, Table 3). When PBL were analysed into lymphocytes, polymorphonuclear cells and monocytes (according to their size and granularity), the reaction of G D A - J / F 7 MoAb was only with monocytes. Thus, by using this
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Fig. 7. FACS analysis of PBL with G D A - J / F 7 MoAb: A, negative control; B, reaction of G D A - J / F 7 MoAb with PBL. The antibody reacted with 85% of monocytes (c) but not with lymphocytes (b) or polymorlahOnuclear cells (d). a: scattergram of the PBL.
133
technique, more than 8507o of the monocytes reacted with the antibody (Fig. 7).
Immunoelectron microscopy (IEM) To ascertain the specificity of GDA-J/F7 MoAb for the SpP, NCOS obtained from an oligospermic donor were tested by immunogold electron microscopy. The results showed the binding of variable numbers of gold particles to about 6007o of NCOS. The cells had the typical morphology of get-
Fig. 8. lmmunoelectron microscopy of the NCOS with GDA-J/F7 MoAb. The two cells (a and c) represent two spermatids at different stages o f differentiation; one before (a) and the other after (c) condensation o f the nuclei. The cell in (c) shows the developing acrosome (curved arrow). (b) and (d) are higher magnifications o f the insets in (a) and (c), respectively, to show the distribution of the gold particles on the surface of the two ceils. (a,c × 9360; b,d × 48,750).
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minal epithelium with some having an acrosome (Fig. 8). In these cells, the gold particles were seen either individually dispersed on the SpP surface or grouped into small clusters. In the negative control, either no binding or only occasional particles were seen (data not shown). Discussion In semen, the NCOS could originate from leucocytes, lining epithelium of the genital tract or germinal epithelium of the testes (Jassim and Festenstein, 1987b). During seminal analysis, the distinction between these different groups of cells is of utmost clinical importance, especially between leucocytes and germ cells. This is because, unlike the SpP, the presence of large numbers of leucocytes in semen is usually associated with genital tract infections; conditions for which further investigations are essential in order to identify the causative pathogen(s) before starting the treatment. The SpP therefore should be properly characterised. If these cells are mistakenly identified as "leucocytes", which often happens with inexperienced seminal analysts, the patients would be subjected to exhaustive microbiological investigations and might receive unnecessary treatment, such as antibiotics. However, the distinction between leucocytes and SlaP has always been difficult and, previously, electron microscopy was the only technique available for their definitive identification (Hughes et al., 1981). Being laborious and time consuming, the EM was later replaced by an immunological approach to identify the different components of the NCOS (Jassim and Festenstein, 1987b). In that particular study, the SpP were found to lack Class I and II MHC antigens (see also Jassim et al., 1989a) as well as other epithelial and leucocytic cell markers. These were considered "negative markers". The only positive marker which reacted with the SpP was the mouse anti-sperm antibody (MASA). MASA, being a xenoantiserum, has several disadvantages, including the limited production of the antibody, necessity for exhaustive absorption with human red blood cells and lymphocytes, variation in the reactivity of the different batches, continuous immunisation, and sacrificing large numbers of mice. Here, as a substitute for MASA, GDA-J/F7 was produced as a specific MoAb for the SlaP, and therefore could be used as an immunological marker for the identification of these cells in semen. The specificity of G D A - J / F 7 MoAb for germ cells was ascertained by (1) its reactivity with NCOS from donors known to produce SpP in their semen, (2) the use of immunoelectron microscopy, which showed the specific binding of the antibody to the germ cells, (3) non- or low reactivity with NCOS made up mainly of leucocytes, (4) non-reactivity with resting or phytohaemagglutinin-stimulated lymphocytes (unpublished data) or with polymorphonuclear cells isolated from peripheral blood. The antibody did, however,
135
TABLE 6 Immunological markers for the distinction between SpP and leucocytes in human semen.
MoAb
Specificity
Leucocytes
SpP
LP34 W6/32 BBM. I GAP 8.3 GDA-J/F7
cytokeratin Class I M H C antigens /1-2 microglobulin leucocytes germ cells and monocytes
+ + + - '
+
'All leucocytes are negative with G D A - J / F 7 MoAb with the exception of monocytes.
react with peripheral blood monocytes, but this should not pose a major problem during seminal analysis. This is because during genital tract infections it is usually the polymorphonuclear cells and/or lymphocytes which predominate rather than the monocytes. Furthermore, by using GDA-J/F7 antibody together with other leucocyte markers (see Table 6), the identification and quantification of the various components of the NCOS can be precisely established. Previously, cytological staining of leucocytes in human semen showed the predominance of lymphocyte subpopulations (Couture et al., 1976; Olsen and Shields, 1984; Zagury et al., 1984). In other studies using MoAbs, the results revealed the presence of polymorphonuclear cells as the predominant type of seminal leucocytes (EI-Demiry et al., 1986a,b; Wolff and Anderson, 1988). This "discrepancy" in results was merely attributed to differences in the methods of detection, cytological as against immunological staining (EIDemiry et al., 1986a; Wolff and Anderson, 1988). However, as the presence of leucocytes in semen, especially in large numbers, may be associated with clinical or subclinical genital tract infections, two more factors may determine the predominance of the leucocyte subpopulation, namely, the chronicity of infection as well as the type of pathogen, whether bacterial or viral. Chronic bacterial and viral infections usually induce lymphocytic responses while acute bacterial infections are frequently associated with the presence of polymorphonuclear cells. In this study, however, there was a tendency towards detecting lymphocytes at higher percentages than polymorphonuclear cells, although the number of normospermic donors was relatively small. As shown here, both FACS and the IIF test could be used for the detection of SpP with GDA-J/F7 MoAb. However, the cells should be tested in suspension as SpP dried onto slides failed to react with the antibody. Similarly, GDA-J/F7 MoAb did not stain germ cells inside the testis when frozen sections were examined. This is in contrast to the reaction of GDA-J/FI
136
MoAb (Jassim and Festenstein, 1987a) which recognises an antigen inside the germ cells when tested as either dried SpP or in frozen sections of testis. The IIF test was useful in showing that GDA-J/F7 MoAb was detecting an antigen on the surface of the SpP, monocytes and mature sperm. This was also confirmed by immunoelectron microscopy. However, the different pattern of immunofluorescence noticed on the SpP surface (this was not due to antigen capping as sodium azide and 4°C incubation had no effect (unpublished data)) may suggest compartmentalisation of the antigen. This may be related to the stages of germ cell differentiation, which was evident from the morphology of the cells. However, as the present study does not rule out completely the possibility of G D A - J / F 7 being a coating antigen, it is premature to speculate further on the biological importance of the antigen in germ cell development. These aspects are currently being investigated together with attempts to characterise the antigen biochemically from both the germ cells and the monocytes. This is essential for the future molecular biology studies on the encoding genes, as well as to show whether the germ cells and monocytes share the same antigens or merely some cross-reacting epitopes.
Acknowledgements The author is indebted to Mr D Auger (Oral Pathology, Dental Institute) for his help with the electron microscopy, Dr P Veys (Department of Haematology, Royal Free Hospital) with the FACS analysis and Dr I Leigh (Department of Dermatology) for providing the LP34 MoAb. The Leverhulme Trust is acknowledged for their financial support and Denny Williams for preparation of the manuscript.
References Bellv¢, A.R. and Moss, S.B. (1983) Monoclonal antibodies as probes of reproductive mechanisms. Biol. Reprod. 28, i--26. Berger, A.E., Davies, J.E. and Cresswell, P. (1981) A human leukocyte antigen identified by a monoclonal antibody. Hum. lmmunol. 3,231--245. Couture, M., Ulstein, M., Leonard, J. and Paulsen, G.A. (1976) Improved staining method for differentiating immature germ cells from white blood cells in human seminal fluid. Andrologia 8, 61 --66. EI-Demiry, M.I.M., Hargreave, T.B., Busuttil, A., James, K. and Chisholm, G.D. (1986a) Identifying leucocytes and leucocyte subpopulations in semen using monoclonal antibody probes. Urology 28, 492--496. EI-Demiry, M.I.M., Young, H., Elton, R.A., Hargreave, T.B., James, K. and Chisholm, G.D. (1986b) Leucocytes in the ejaculate from fertile and infertile man. Br. J. Urol. 58, 715--720. Hoffman, R.A., Kung, P.C., 14ansen, W.P. and Goldstein, G. (1980) Simple and rapid measurement of human T lymphocytes and their subclasses in peripheral blood. Proc. Natl. Acad. Sci. U.S.A. 77, 4914--4917. Hughes, L., Ryder, T.A., McKenzie, M.L., Pryse-Davies, J., Stedronska, J. and Hendry, W.F. (1981) The use of transmission electron microscopy to study nonspermatozoal cells in semen. In: Oligozoospermia, Recent Progress in Andrology (Frajese, G., Hafez, E.S.E., Conti, C. and Fabbrini, F., eds.), pp. 65--75. Raven Press, New York.
137 Jassim, A. and Festenstein, H. (1987a) Molecular dissection of human testicular germ cell differentiation with monoclonal antibodies. J. Reprod. Immunol. 12, 173--189. Jassim, A. and Festenstein, H. (1987b) Immunological and morphological characterisation of nucleated cells other than sperm in semen of oligospermic donors. J. Reprod. Immunol. 1 I, 77--89. Jassim, A. and Festenstein, H. (1988) Identification of spermatids with aprotruded tails in human semen by monoclonal antibodies and electron microscopy. In: Molecular and Cellular Endocrinology of the Testis (Cooke, B.A. and Sharpe, R., eds.), pp. 203--208. Raven Press. Jassim, A., Oliver, R.T.D., Blandy, J.P. and Festenstein, H. (1990) Agenesis of human sperm tail revealed with GDA-J/F3 monoclonal antibody. In: Treatment of Infertility (Boutaleb, Y. and Gzouli, A., eds.), Parthenon Publishing (in press). Jassim, A., Oilier, W., Payne, A., Biro, A., Oliver, R.T.D. and Festenstein, H. (1989a) Analysis of HLA antigens on germ cells in human semen. Eur. J. Immunol. 19, 1215--1220. Jassim, A., Payne, A., Oliver, R.T.D. and Festenstein, H. (1989b) Characterisation of human male germ cells with monoclonal antibodies against mitogen-activated lymphocyte antigens and CD markers. Tissue Antigens 33,106 Lampson, L.A. and Levy, R. (1980) Two populations of la-like molecules on a human B cell line. J. lmmunol. 125,293--299. Mandelbanm, S.L., Diamond, M.P. and DeCherney, A.H. (1986) Immunology of the sperm-egg interacdon. J. in Vitro Fertil. Embryo. Transfer 3,279--283. Moore, H.D., Hartman, T.D., Bye, A.P., Lutjen, P., DeWitt, M. and Trounson, A.O. (1987) Monoclonai antibody against sperm antigen M 95,000 inhibits attachment of human spermatozoa to the zona pellucida. J. Reprod. Immunol. 11,157--166. Oisen, G.P. and Shields, J.W. (1984) Seminal lymphocytes, plasma and Aids. Nature 309, 116--117. Parham, P., Barnstable, C.J. and Bodmer, W.F, (1979) Use of a monoclonal antibody (W6/32) in strucrural studies of HLA-A, B, C antigens. J. Immunol. 123,342--349. Ruiz-Cabello, F., Nevot, M.A.L., 6arrido, A. and Garrido, F. (1987) A study of GRMI monoclonal antibody that reacts with natural killer cells and granulocytes. Nat. lmmun. Cell Growth Reg. 6, 99-108. Salonen, 1. and Kallajoki, M. (1987) Monoclonal antibody against human sperm acrosome inhibits sperm penetration of zona-free hamster eggs. Int. J. Androl. 10, 731--739. Ugolini, V., Nunez, G., Smith, R.G., Stasmy, P. and Capra, D. (1980) lnititai characterisation of monoclonai antibodies against human monocytes. Proc. Natl. Acad. Sci. U.S.A. 77, 6764--6768. Witkin, S.S. and Goldstein, M. {1988) Reduced levels of T suppressor/cytotoxic lymphocytes in semen from vasovasostomised men: relationship to sperm auto-antibodies. J. Reprod. Immunol. 14, 283-290. Wolff, H. and Anderson, D.J. (1988) lmmunohistologic characterisation and quantitation of leukocyte subpopulations in human semen. Fertil. Steril. 49, 497--504. Yan, Y.C., Wang, L.F. and Koide, S.S. (1987) Properties of a monoclonal antibody interacting with human sperm. Arch. Androl. 18,245--254. Zagury, D., Bernard, J., Leibowitch, J., Safai, B., Groopman, J.E., Feldman, M., Sarngadharan, M.G. and Gallo, R.C. (1984) HTLV-lll in cells cultured from semen of two patients with AIDS. Science 226, 449--45 I.
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Elsevier Scientific Publishers Ireland Ltd.
JRI 00674
G D A - J / F 7 monoclonal antibody: A new marker for sperm cell precursors in human semen A. J a s s i m Department of Immunology, London Hospital Medical College, Turner Street, London E1 2AD (U.K.) (Accepted for publication 16 March 1990)
Summary Germ cells isolated from semen of oligospermic human donors were found to react with GDA-J/F7 monoclonal antibody (MoAb). Their reactions with this antibody were demonstrated by using fluorescein activated cell sorter (FACS) analysis and indirect immunofluorescence (IIF) test. In the IIF test, the MoAb recognised an antigen on the surface of the sperm cell precursors (SpP) as well as on mature spermatozoa. The specificity of the antibody reaction with the SpP was further confirmed by immunoelectron microscopy. The MoAb did not react with peripheral blood lymphocytes or polymorphonuclear cells but did show cross-reactivity with monocytes. This antibody therefore provides the first marker for the SpP and could be used as a probe for their distinction from leucocytes. This could have clinical application in seminal analysis. Key words: sperm cell precursors; surface antigen; monoclonal antibody;
human semen.
Introduction The development o f monoclonal antibody technology has greatly helped in the characterisation and identification o f a large number o f cellular antigens. Some o f these antigens have been found to be specific for a particular tissue or a group o f cells; consequently the antibodies can be used as markers 0165-0378/90/$03.50 © 1990Elsevier Scientific Publishers Ireland Ltd. Pubhshed and Printed in Ireland
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for their identification, or even the classification of one group of cells into various subsets. In the field of reproduction, monoclonal antibodies (MoAbs) have been extensively used to study various biological phenomena, including germ cell differentiation (Jassim and Festenstein, 1987a), fertilisation and sperm-egg interactions (Mandelbaum et al., 1986; Salonen and Kallajoki, 1987; Moore et al., 1987), detection of abnormal germ cells in human semen (Jassim and Festenstein, 1988; Jassim et al., 1990) and others (Bellve and Moss, 1983; Yan et al., 1987). With few exceptions, most studies on human germ cells have concentrated on the use of MoAbs to analyse antigens on mature sperm (see Jassim and Festenstein, 1987a). Thus, immunological markers for SpP have been lacking. Consequently, in previous studies on the cellular analysis of human semen (EI-Demiry et al., 1986a,b; Wolff and Anderson, 1988; Witkin and Goldstein, 1988) the MoAbs used were only those against leucocytes and their various subsets, i.e. no marker for the germ cell was used. In this report a MoAb for the identification of the SpP is described, together with its potential clinical application in seminal analysis as a probe for the distinction between these cells and the leucocytes. Materials and Methods
Monoclonai antibodies (MoAbs) The MoAbs used in this study are listed in Table 1. All the antibodies which were used as supernatants were of mouse origin. The MoAbs against leucocytes and their different subsets (Table 1), as shown previously (Jassim et al., 1989a) and in this study (see Results), show no cross reaction with germ cells or subcellular particles (cf. Wolff and Anderson, 1988).
TABLE I MoAbs used in the characterisation of NCOS.
MoAb
Reference
LP34 G A P 8.3 GRMI 63D3 OKT3 W6/32 L243 GDA-J,.F7
(I. Leigh) Berger et al., 1981 Ruiz-Cabello et al., 1987 Ugolini et al., 1980 H o f f m a n et al., 1980 Parham et al., 1979 Lampson and L e w , 1980 Jassim and Festenstein 1987a
125
Sperm donors The oligospermic donors used in this study were medical students who were previously shown to produce large numbers of nucleated cells other than sperm fNCOS) and low counts of sperm in their semen (Jassim and Festenstein, 1987b). In addition, 7 normospermic donors whose semen contained leucocytes (as characterised herein) were also included.
Separation of NCOS and peripheral blood leucocytes (PBL) The method for the separation of the NCOS from human semen and PBL from peripheral blood has been described previously (Jassim and Festenstein, 1987b). Basically, the technique involves the use of ficoll/triosil as the density gradient. The rate of NCOS recovery by using this method exceeds 90070 and the contamination with sperm is less than 10070. This has been demonstrated previously (Jassim and Festenstein, 1987b; Jassim et al., 1989a) as well as in the present study (see Figs. 1 and 2). The use of viable cells reduces the cross reaction of MoAbs with unrelated cytoplasmic antigens inside the germ cells (Jassim et al., 1989b).
Indirect immunofluorescence (IIF) test and FA CS analysis Two million washed cells (NCOS, PBL or sperm) were incubated with 100 /al of MoAb for 1 h at 37 °C. The cells were washed twice with phosphate buffered saline (PBS) and then incubated with 4/A of rabbit F(ab) 2 antimouse immunoglobulin conjugated to fluorescein isothiocyanate (Sigma) for 30 rain at 20 °C. The samples were finally washed 3 times with PBS and examined by using a fluorescence microscope a n d / o r subjected to FACS analysis. Flow cytometry was performed using a Coulter Epics C to measure excitation at 488 nm on a 10,000 cell count. Lymphocyte, monocyte and granulocyte cell populations were gated separately. Background fluorescence was set in each experiment by assaying cells incubated with the fluorescein-conjugated antibody only, i.e. without the primary MoAb. For testing LP34 MoAb, which recognises cytokeratin intermediate filaments, the cells were first fixed with 50:50 methanol/acetone before the addition of the antibody.
Immunoelectron microscopy Four million washed NCOS obtained from an oligospermic donor were incubated with 200/al of GDA-J/F7 MoAb for 1 h at 37 °C. The cells were washed twice with PBS and then incubated with 10 ~I of colloidal gold goat anti-mouse immunoglobulin (size 5 nm, Janssen) at 37°C for 50 min with frequent agitation. The NCOS were finally washed 3 times with PBS and fixed with 2°70 gluteraldehyde in PBS. Further processing of the samples for
126
O Fig. I. Indirect immunofluorescence test of NCOS from an oligospermic donor ~ith GDA-J F7 MoAb: a, phase contrast; b, fluorescence microscopy. Note the surface labelling of the cells v, ith the antibod.~ and variation in the pattern of the immunofluorescence staining, dotted (curxed arrow); circular 0ong arrow); polar (arrow head) or interrupted circle (small arrowl. ( × 210).
Fig. 2. Indirect immunofluorescence test of NCOS from an oligospermic donor with W6/32 (a,b) and G A P 8.3 (c,d). The two antibodies failed to react with germ cells (cf. Fig. I) but stained occasional cells (arrows) indicating their origin from leucocytes. These cells showed the morphology of lymphocytes. (a,b) x 330; (c,d} x 210.
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electron microscopy was as described previously (Jassim and Festenstein, 1987b). Results
Reaction o f G D A - J / F 7 M o A b with germ cells in semen o f oligospermic donors Following their separation on a ficoll density gradient, the NCOS were tested with GDA-J/F7 MoAb using the IIF technique. The antibody produced strong immunofluorescence around the surface of 90--100°70 of the NCOS. The pattern of cell staining was variable, showing dotted, circular, polar or interrupted circle immunofluorescence (Fig. 1). This was noticed on cells which differed in size as well as internal structures. Using the IIF technique, the NCOS were also tested with a panel of tissuespecific MoAbs. In these experiments, the anti-leucocyte (and their subsets), anti-epithelial cells, anti-HLA Class I and anti-HLA-DR MoAbs failed to react with the NCOS. However, following the screening of a large number of NCOS, occasional cells were found positive with anti-leucocyte, anti-lymphocyte and anti-MHC Class I MoAbs. These cells showed the morphology of leucocytes and were probably lymphocytes (Fig. 2). Similar results were obtained by using FACS analysis where all the
L :L L .
L L
Fig. 3. FACS analysis of the NCOS from an oligospermic donor with tissue specific MoAbs: a, RPMI; b, LP34; c, GAP 8.3; d, GRMI; e, 63D3; f, OKT3; g, W6/32; h, L243; i, GDA-J/F7. The failure of the antiepithelial, anti-leucocytes and their subsets and anti-MHC Class I and HLA-DR MoAbs to react with the NCOS indicates their germinal origin. GDA-J/F7 MoAb reacted strongly with these cells (see also Table 2).
C
128 TABLE 2 FACS analysis of NCOS from oligospermic donors with tissue-specific MoAbs. The reactivity of the anti-epithelial, anti-leucocyte and their subsets and anti-MHC Class I and HLA-DR MoAbs with less than !% NCOS indicates their germ cell origin (see Fig. 3). MoAb
Specificity
% of positive cell
RPMI LP34 G A P 8.3 GRMI 63D3 OKT3 W6/32 L243 GDA-J/F7
negative control epithelial cells (keratin) leucocytes granulocytes and NK cells monocytes T lymphocytes Class I M H C antigens HLA-DR germ cells and monocytes
0.10 0.18 0.34 0.68 0.38 0.6 I 0.58 0.32 75.01
A
L~
L L L
B
a
I
i
b
C
dL
C
......
d
Fig. 4. FACS analysis of the reactions of G D A - J / F 7 and anti-leucocyte M o A b s with NCOS (A) and PBL (B) of an oligospermic donor: a, G A P 8.3; b, GRMI; c, OKT3; d, 63D3; e, GDA-J/FT. Note the strong reaction of the NCOS but not the PBL with G D A - J / F 7 MoAb. The NCOS, unlike PBL, did not react with the anti-leucocyte (and their subsets) M o A b s (see also Table 3).
129
MoAbs reacted with less than 1°70 of NCOS except GDA-J/F7. This antibody reacted with 75--100070 of NCOS. The results of one representative experiment using FACS analysis are shown in Table 2 and Fig. 3. In this particular experiment, the GDA-J/F7 MoAb reacted with 75% of the NCOS (Table 2). Figure 4 and Table 3 show a comparison of the reactivity of the MoAbs with the NCOS and PBL of the same donor. The results revealed the reaction of GDA-J/F7 with 78°70 of the NCOS while the anti-leucocyte MoAb (and antibodies against their various subsets) reacted with less than 1°70 of NCOS, i.e. similar to that of the negative control (data not shown). This was in contrast to their reactions with the PBL where the G A P 8.3 MoAb reacted with 99°70 of the cells while the GDA-J/F7 showed a reaction of 6°70 only (this, as discussed later, was against monocytes). The MoAbs against the leucocyte subpopulations reacted with the PBL in percentages corresponding to their specificities.
Reaction of GDA-J/F7 MoAb with leucocytes in semen o f normospermic donors The leucocytes in the semen of the normospermic donors were identified by using tissue specific MoAbs and anti-MHC Class I and HLA-DR antibodies as additional markers. The cells were tested by FACS analysis and IIF microscopy. Using the IIF technique, the anti-leucocyte and anti-MHC Class I MoAbs reacted with 82--95°70 of NCOS (Table 4). The GDA-J/F7 MoAb, on the other hand, showed a reaction with 7--130/0 of NCOS. In these donors, there was a tendency for the OKT3 antibody to react with higher percentages of NCOS than GRMI. TABLE3 FACS analysis of NCOS and PBL of an oligospermic donor with G D A - J / F 7 and anti-leucocyte MoAbs. Note the strong reaction o f the NCOS but not the PBL with G D A - J / F 7 . The NCOS, unlike the PBL, did not react with the anti-leucocyte (and their subsets) M o A b s (see Fig. 4). The negative control was less than ! % (data not shown). MOAb
NCOS
PBL
G A P 8.3' GRMI OKT3 63D3 GDA-J/F7
0.47 b 0.41 0.28 0.37 78.00
99.7 21.0 58.0 2.0 6.4
' For specificity of the MoAbs, see Table 2. b Numbers represent percentage of positive cells.
130
TABLE 4 Characterisation of NCOS from 7 normosperic donors with MoAbs by |IF microscopy. The reaction of the anti-leucocyte and anti-MHC Class I MoAbs with the NCOS indicate that the majority of the cells were leucoc}ae in origin. G D A - J / F 7 showed minimal reactivity. MoAb
RPMP LP34 GAP 8.3 GRMI 63 D3 OKT3 W6,, 32 L243 GDA-J ,"F7
NCOS from donor A
B
C
D
E
F
G
It 3 90 17 25 50 95 25 8
0 2 85 20 23 44 90 20 7
I I 82 45 30 I0 87 30 13
2 2 95 18 28 40 91 30 I0
2 I 87 21 18 50 93 25 8
1 2 90 15 28 39 88 30 7
2 I 95 18 20 45 90 26 7
For specificity of the MoAbs, see Table 2. ° Numbers represent percentage of positive cells.
d
L,~
g
,.~
~
e
h
~_
_
r
Fig. 5. FACS analysis of the NCOS from a normospermic donor with tissue specific MoAbs: a, RPMI; b, LP34; c, GAP 8.3; d, GRMI; e, 63D3; f, OKT3; g, W6/32; h, L243; l, GDA-J/F7. The reactions of the anti-leucocytes and their subsets and anti-MHC Class I and HLA-DR MoAbs indicate that the majority of the NCOS were leucocytes in origin. G D A - J / F 7 MoAb showed Iov, reactivity (see also Table 5).
131 TABLE 5 FACS analysis o f NCOS from normospermic donors with tissue-specific MoAbs. The reactivity of the anti-leucocytes and their subsets and anti-MHC Class I and HLA-DR MoAbs with the NCOS indicates that the majority of the NCOS were leucocytes (see Fig. 5). MoAB
Specificity
% of positive cell
RPMI LP34 G A P 8.3 GRM I 63D3 OKT3 W6/32 L243 GDA-J/F7
negative control epithelialcells(keratin) leucocytes granulocytes and N K cells monocytes T lymphocytes Class I MHC antigens HLA-DR germ cells and monocytes
0,86 0.93 90.01 16.46 21.42 6.50 63.40 16.20 8. l0
The FACS analysis showed results comparable to those obtained by IIF microscopy. Thus, the anti-leucocyte and anti-Class I HLA MoAbs reacted with 60--95070 of the NCOS, while the antibodies against the various leucocyte subsets recognised cells in percentages corresponding to their specificities. The anti-epithelial LP34 MoAb reacted with less than 1o70 of NCOS. The GDA-J/F7 MoAb in all these experiments showed a reaction with 5-15o70 of NCOS. The results of one such experiment using FACS technique are shown in Fig. 5 and Table 5. In contrast to the reaction of the anti-leucocyte MoAb with 90°70 of NCOS, the GDA-J/F7 antibody reacted with only 8070 of the cells (Table 5).
Fig. 6. Indirect immunofluorescence test of sperm with GDA-J/F7 MoAb: a, phase contrast; b, fluores-
132
Reaction o f G D A - J / F 7 M o A b with sperm Ejaculated sperm from normospermic and oligospermic donors were tested with GDA-J/F7 MoAb using the IIF test. The antibody produced patchy fluorescence along the entire surface of 90--100°/0 sperm of both groups of donors (Fig. 6). Reaction o f G D A - J / F 7 M o A b with dried germ cells Germ cells obtained from seminal samples of oligospermic donors were dried onto slides. These cells were tested with GDA-J/F7 MoAb using the IIF technique. This test was also used to stain frozen sections of normal adult testes. The results showed the failure of the antibody to react with the dried germ cells. Reaction o f G D A - J / F 7 M o A b with PBL By using the IIF technique, G D A - J / F 7 MoAb stained 3--10°70 of PBL. The cells showed dotted immunofluorescence around their surface. Similar results were obtained when the samples were tested using the FACS technique (Fig. 4, Table 3). When PBL were analysed into lymphocytes, polymorphonuclear cells and monocytes (according to their size and granularity), the reaction of G D A - J / F 7 MoAb was only with monocytes. Thus, by using this
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.
.
.
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.
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.
.
.
.
.
.
.
.
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b
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Fig. 7. FACS analysis of PBL with G D A - J / F 7 MoAb: A, negative control; B, reaction of G D A - J / F 7 MoAb with PBL. The antibody reacted with 85% of monocytes (c) but not with lymphocytes (b) or polymorlahOnuclear cells (d). a: scattergram of the PBL.
133
technique, more than 8507o of the monocytes reacted with the antibody (Fig. 7).
Immunoelectron microscopy (IEM) To ascertain the specificity of GDA-J/F7 MoAb for the SpP, NCOS obtained from an oligospermic donor were tested by immunogold electron microscopy. The results showed the binding of variable numbers of gold particles to about 6007o of NCOS. The cells had the typical morphology of get-
Fig. 8. lmmunoelectron microscopy of the NCOS with GDA-J/F7 MoAb. The two cells (a and c) represent two spermatids at different stages o f differentiation; one before (a) and the other after (c) condensation o f the nuclei. The cell in (c) shows the developing acrosome (curved arrow). (b) and (d) are higher magnifications o f the insets in (a) and (c), respectively, to show the distribution of the gold particles on the surface of the two ceils. (a,c × 9360; b,d × 48,750).
134
minal epithelium with some having an acrosome (Fig. 8). In these cells, the gold particles were seen either individually dispersed on the SpP surface or grouped into small clusters. In the negative control, either no binding or only occasional particles were seen (data not shown). Discussion In semen, the NCOS could originate from leucocytes, lining epithelium of the genital tract or germinal epithelium of the testes (Jassim and Festenstein, 1987b). During seminal analysis, the distinction between these different groups of cells is of utmost clinical importance, especially between leucocytes and germ cells. This is because, unlike the SpP, the presence of large numbers of leucocytes in semen is usually associated with genital tract infections; conditions for which further investigations are essential in order to identify the causative pathogen(s) before starting the treatment. The SpP therefore should be properly characterised. If these cells are mistakenly identified as "leucocytes", which often happens with inexperienced seminal analysts, the patients would be subjected to exhaustive microbiological investigations and might receive unnecessary treatment, such as antibiotics. However, the distinction between leucocytes and SlaP has always been difficult and, previously, electron microscopy was the only technique available for their definitive identification (Hughes et al., 1981). Being laborious and time consuming, the EM was later replaced by an immunological approach to identify the different components of the NCOS (Jassim and Festenstein, 1987b). In that particular study, the SpP were found to lack Class I and II MHC antigens (see also Jassim et al., 1989a) as well as other epithelial and leucocytic cell markers. These were considered "negative markers". The only positive marker which reacted with the SpP was the mouse anti-sperm antibody (MASA). MASA, being a xenoantiserum, has several disadvantages, including the limited production of the antibody, necessity for exhaustive absorption with human red blood cells and lymphocytes, variation in the reactivity of the different batches, continuous immunisation, and sacrificing large numbers of mice. Here, as a substitute for MASA, GDA-J/F7 was produced as a specific MoAb for the SlaP, and therefore could be used as an immunological marker for the identification of these cells in semen. The specificity of G D A - J / F 7 MoAb for germ cells was ascertained by (1) its reactivity with NCOS from donors known to produce SpP in their semen, (2) the use of immunoelectron microscopy, which showed the specific binding of the antibody to the germ cells, (3) non- or low reactivity with NCOS made up mainly of leucocytes, (4) non-reactivity with resting or phytohaemagglutinin-stimulated lymphocytes (unpublished data) or with polymorphonuclear cells isolated from peripheral blood. The antibody did, however,
135
TABLE 6 Immunological markers for the distinction between SpP and leucocytes in human semen.
MoAb
Specificity
Leucocytes
SpP
LP34 W6/32 BBM. I GAP 8.3 GDA-J/F7
cytokeratin Class I M H C antigens /1-2 microglobulin leucocytes germ cells and monocytes
+ + + - '
+
'All leucocytes are negative with G D A - J / F 7 MoAb with the exception of monocytes.
react with peripheral blood monocytes, but this should not pose a major problem during seminal analysis. This is because during genital tract infections it is usually the polymorphonuclear cells and/or lymphocytes which predominate rather than the monocytes. Furthermore, by using GDA-J/F7 antibody together with other leucocyte markers (see Table 6), the identification and quantification of the various components of the NCOS can be precisely established. Previously, cytological staining of leucocytes in human semen showed the predominance of lymphocyte subpopulations (Couture et al., 1976; Olsen and Shields, 1984; Zagury et al., 1984). In other studies using MoAbs, the results revealed the presence of polymorphonuclear cells as the predominant type of seminal leucocytes (EI-Demiry et al., 1986a,b; Wolff and Anderson, 1988). This "discrepancy" in results was merely attributed to differences in the methods of detection, cytological as against immunological staining (EIDemiry et al., 1986a; Wolff and Anderson, 1988). However, as the presence of leucocytes in semen, especially in large numbers, may be associated with clinical or subclinical genital tract infections, two more factors may determine the predominance of the leucocyte subpopulation, namely, the chronicity of infection as well as the type of pathogen, whether bacterial or viral. Chronic bacterial and viral infections usually induce lymphocytic responses while acute bacterial infections are frequently associated with the presence of polymorphonuclear cells. In this study, however, there was a tendency towards detecting lymphocytes at higher percentages than polymorphonuclear cells, although the number of normospermic donors was relatively small. As shown here, both FACS and the IIF test could be used for the detection of SpP with GDA-J/F7 MoAb. However, the cells should be tested in suspension as SpP dried onto slides failed to react with the antibody. Similarly, GDA-J/F7 MoAb did not stain germ cells inside the testis when frozen sections were examined. This is in contrast to the reaction of GDA-J/FI
136
MoAb (Jassim and Festenstein, 1987a) which recognises an antigen inside the germ cells when tested as either dried SpP or in frozen sections of testis. The IIF test was useful in showing that GDA-J/F7 MoAb was detecting an antigen on the surface of the SpP, monocytes and mature sperm. This was also confirmed by immunoelectron microscopy. However, the different pattern of immunofluorescence noticed on the SpP surface (this was not due to antigen capping as sodium azide and 4°C incubation had no effect (unpublished data)) may suggest compartmentalisation of the antigen. This may be related to the stages of germ cell differentiation, which was evident from the morphology of the cells. However, as the present study does not rule out completely the possibility of G D A - J / F 7 being a coating antigen, it is premature to speculate further on the biological importance of the antigen in germ cell development. These aspects are currently being investigated together with attempts to characterise the antigen biochemically from both the germ cells and the monocytes. This is essential for the future molecular biology studies on the encoding genes, as well as to show whether the germ cells and monocytes share the same antigens or merely some cross-reacting epitopes.
Acknowledgements The author is indebted to Mr D Auger (Oral Pathology, Dental Institute) for his help with the electron microscopy, Dr P Veys (Department of Haematology, Royal Free Hospital) with the FACS analysis and Dr I Leigh (Department of Dermatology) for providing the LP34 MoAb. The Leverhulme Trust is acknowledged for their financial support and Denny Williams for preparation of the manuscript.
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137 Jassim, A. and Festenstein, H. (1987a) Molecular dissection of human testicular germ cell differentiation with monoclonal antibodies. J. Reprod. Immunol. 12, 173--189. Jassim, A. and Festenstein, H. (1987b) Immunological and morphological characterisation of nucleated cells other than sperm in semen of oligospermic donors. J. Reprod. Immunol. 1 I, 77--89. Jassim, A. and Festenstein, H. (1988) Identification of spermatids with aprotruded tails in human semen by monoclonal antibodies and electron microscopy. In: Molecular and Cellular Endocrinology of the Testis (Cooke, B.A. and Sharpe, R., eds.), pp. 203--208. Raven Press. Jassim, A., Oliver, R.T.D., Blandy, J.P. and Festenstein, H. (1990) Agenesis of human sperm tail revealed with GDA-J/F3 monoclonal antibody. In: Treatment of Infertility (Boutaleb, Y. and Gzouli, A., eds.), Parthenon Publishing (in press). Jassim, A., Oilier, W., Payne, A., Biro, A., Oliver, R.T.D. and Festenstein, H. (1989a) Analysis of HLA antigens on germ cells in human semen. Eur. J. Immunol. 19, 1215--1220. Jassim, A., Payne, A., Oliver, R.T.D. and Festenstein, H. (1989b) Characterisation of human male germ cells with monoclonal antibodies against mitogen-activated lymphocyte antigens and CD markers. Tissue Antigens 33,106 Lampson, L.A. and Levy, R. (1980) Two populations of la-like molecules on a human B cell line. J. lmmunol. 125,293--299. Mandelbanm, S.L., Diamond, M.P. and DeCherney, A.H. (1986) Immunology of the sperm-egg interacdon. J. in Vitro Fertil. Embryo. Transfer 3,279--283. Moore, H.D., Hartman, T.D., Bye, A.P., Lutjen, P., DeWitt, M. and Trounson, A.O. (1987) Monoclonai antibody against sperm antigen M 95,000 inhibits attachment of human spermatozoa to the zona pellucida. J. Reprod. Immunol. 11,157--166. Oisen, G.P. and Shields, J.W. (1984) Seminal lymphocytes, plasma and Aids. Nature 309, 116--117. Parham, P., Barnstable, C.J. and Bodmer, W.F, (1979) Use of a monoclonal antibody (W6/32) in strucrural studies of HLA-A, B, C antigens. J. Immunol. 123,342--349. Ruiz-Cabello, F., Nevot, M.A.L., 6arrido, A. and Garrido, F. (1987) A study of GRMI monoclonal antibody that reacts with natural killer cells and granulocytes. Nat. lmmun. Cell Growth Reg. 6, 99-108. Salonen, 1. and Kallajoki, M. (1987) Monoclonal antibody against human sperm acrosome inhibits sperm penetration of zona-free hamster eggs. Int. J. Androl. 10, 731--739. Ugolini, V., Nunez, G., Smith, R.G., Stasmy, P. and Capra, D. (1980) lnititai characterisation of monoclonai antibodies against human monocytes. Proc. Natl. Acad. Sci. U.S.A. 77, 6764--6768. Witkin, S.S. and Goldstein, M. {1988) Reduced levels of T suppressor/cytotoxic lymphocytes in semen from vasovasostomised men: relationship to sperm auto-antibodies. J. Reprod. Immunol. 14, 283-290. Wolff, H. and Anderson, D.J. (1988) lmmunohistologic characterisation and quantitation of leukocyte subpopulations in human semen. Fertil. Steril. 49, 497--504. Yan, Y.C., Wang, L.F. and Koide, S.S. (1987) Properties of a monoclonal antibody interacting with human sperm. Arch. Androl. 18,245--254. Zagury, D., Bernard, J., Leibowitch, J., Safai, B., Groopman, J.E., Feldman, M., Sarngadharan, M.G. and Gallo, R.C. (1984) HTLV-lll in cells cultured from semen of two patients with AIDS. Science 226, 449--45 I.
