Journal of Reproductive Immunology, 4 (1982) 315-324 Elsevier Biomedical Press
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An objective sperm cytotoxicity assay for male-specific antisera based on ATP levels of unlysed cells" Application to assay of H-Y antigen Pierre S. Tung, Robert E. Gore-Langton and Irving B. Fritz Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Canada M5G 1L6 (Received 19 March 1982; accepted 19 April 1982)
We correlated the decrease in levels of ATP in spermatozoa with the extent of cytotoxicity elicited by antibodies against antigenic components on sperm. In the presence of concentrations of complement which did not cause cytolysis or influence the ATP content of epididymal sperm, addition of heat-inactivated sera from non-immunized mice, rats or rabbits did not result in sperm cytolysis or a fall in ATP content. In contrast, addition of rabbit anti-rat spermatocyte sera, which has previously been shown to react with rat spermatozoa (Tung, P.S. and Fritz, I.B. (1978) Dev. Biol. 64, 297-315), did cause sperm cytolysis and a decrease in ATP content. The titre of this antiserum for 50% cytolysis was between 1 : 128 and 1 : 256, as determined by the fall in ATP content. Using these criteria, we examined the cytotoxicity against sperm of different samples of anti H-Y sera. We examined the influences of monoclonal antibody against H-Y, mouse H-Y antisera and rat H-Y antisera raised in inbred females immunized with spleen cells from males of the same strains. In all cases, anti-H-Y lowered ATP levels and lysed sperm with a cytotoxic titre between 1:8 and 1 : 16. Measurements of the decrease in ATP content in sperm have been shown to provide an objective and reliable estimate of the percentage of spermatozoa lysed by H-Y antisera. Cytotoxic activity of H-Y antisera was removed by absorption with spleen cells from male mice but not by absorption with spleen cells from female mice.
Introduction T h e h y p o t h e s i s h a s b e e n a d v a n c e d t h a t H - Y a n t i g e n is i m p l i c a t e d i n sex d e termination, causing the indifferent mammalian gonad to differentiate into a testis ( O h n o , 1979). L e v e l s o f H - Y a n t i g e n h a v e b e e n d e t e r m i n e d p r i m a r i l y b y s e r o l o g i c a l a s s a y s , b a s e d u p o n i n t e r a c t i o n s o f a n t i - H - Y a n t i b o d y w i t h m a l e cells, p a r t i c u l a r l y spermatozoa. Most assays measure complement-dependent Cytotoxicity, assessed u s u a l l y b y s u p r a v i t a l s t a i n i n g o f s p e r m w i t h t r y p a n b l u e ( G o l d b e r g et al., 1971), o r r o s e t t e f o r m a t i o n b e t w e e n s p e r m a n d s h e e p r e d b l o o d cells c o m p l e x e d t o p r o t e i n A ( P A - S R B C ) ( K o o a n d G o l d b e r g , 1978). B o t h o f t h e s e m e t h o d s r e q u i r e m i c r o s c o p i c identification and counting of stained spermatozoa or rosettes, and are semi-quantitative at best. Reasoning that cellular energy production would decrease after cytolysis, S u o m i n e n et al. ( 1 9 8 0 ) d e v e l o p e d a n o b j e c t i v e c y t o t o x i c i t y a s s a y d e p e n d e n t u p o n t h e 0165-0378/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
316 analysis of ATP levels. They showed that antibodies cytotoxic to human sperm could be evaluated quantitatively by analyzing ATP levels in sperm incubated with antibodies in the presence and absence of active complement (Suominen et al., 1980). In this communication, we describe modifications of Suominen's method and present data showing the applicability of these procedures for the objective analysis of levels of anti-H-Y antibody.
Materials and Methods
Cell preparation Spleen cells were isolated from outbred Wistar Crl: (WI)BR rats (Canadian Breeding Farm and Laboratories Ltd., Montreal), inbred Lewis/Wistar rats (Biological Associates, Bethesda, Md.) or C57BL/6J mice (Jackson Laboratories, Bar Harbor, Mass.). For sperm preparation, epididymides from outbred Wistar rats were dissected free of fat tissue, and were placed in chilled (4°C) Tyrode buffer supplemented with 10 mM sodium lactate (Gibco) and 2.5 m g / m l of bovine serum albumin (Fraction V, Sigma). The final solution was adjusted to pH 7.4 with 0.1 N acetic acid. The organs were then sliced with a razor-blade into 0.5 cm segments, and gently vortexed to release spermatozoa. Tissues were allowed to settle for 2 min on ice after which the supernatant fraction was filtered (Nytex, 180 mesh/inch), and centrifuged at 4°C, 2500 X g for 5 min. The pellet containing spermatozoa was resuspended in an equal volume of the same modified Tyrode buffer, and kept on ice until use.
Preparation of antisera Rat H-Y antiserum was produced in inbred Lewis/Wistar female rats by repeated intraperitoneal (i.p.) inoculation twice weekly with male spleen cells prepared from the same strain. Immediately following injection of spleen cells, complete Freund's adjuvant (Merck) was subcutaneously administered at six separate sites at a dose of 2.0 m l / r a t for the first to third injection and thereafter the adjuvant was replaced by incomplete Freund's adjuvant (Merck). The rats were bled by aortic puncture 2 weeks after the 8th injection, and aliquots of serum were stored at - 3 2 ° C . Negative control serum was similarly produced in nulliparous females injected with Freund's adjuvant and phosphate-buffered saline (PBS) instead of spleen cells. A further cytotoxic antiserum against various germ cell antigens was raised in a male Albino rabbit by bi-weekly intravenous injections of 4 X 10 7 rat pachytene spermatocytes as described previously (Tung and Fritz, 1978). Mouse anti-H-Y serum was raised in C57BL/6J females by i.p. injection of 50 X 10 6 spleen cells from male mice in each of 7 or 8 successive injections at weekly intervals. Control serum was prepared from non-immunized nulliparous females of the same age. Serum was prepared after collection of blood from the brachial artery. For cytotoxicity tests, all sera used were heat-inactivated at 56°C for 30 min.
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Monoclonal antibody against H-Y antigen was generously provided by Dr. Gloria Koo of the Sloan Kettering Memorial Center, New York.
Absorption of antisera Male or female spleen cells from outbred Wistar rats were used for absorption. This was carried out by adding 100 t~l of 1 : 8 diluted antiserum to a pellet of 6 × l07 spleen cells. After being gently vortexed and kept on ice for 40 min, the supernatant fraction was collected after centrifugation at 6000 X g for 10 min. Cytotoxicity test Spermatozoa (5 X 106), suspended in modified Tyrode's buffer, were pelleted in a 1 ml plastic centrifuge tube at 2500 X g for 5 min. The supernatant fraction was aspirated with a fine-tip Pasteur pipette, and 50 /~1 anti-serum was immediately added to the sperm. This was followed by addition of 50/~1 of reconstituted guinea pig complement (Gibco, diluted with modified Tyrode's solution to 1:20). The mixture was gently resuspended, stoppered and incubated at 32°C for 1 h. To terminate the reaction, each sample was injected into 300/~1 of boiling water, and maintained in a boiling water bath for 10 min. Samples were then transferred to an ice bath or freezer, and stored until needed. A TP assay ATP standard solutions, and the reconstituted luciferase-luciferin reagent (Sigma) were prepared daily, and kept On ice during the experiment. Sperm samples were thawed, and diluted with 2.6 ml modified Tris-HC1 buffer (0.025 M, pH 7.8, containing 4 mM MgSO4) in a plastic scintillation sac supported with a translucent holder (Webster et al., 1980). Reconstituted luciferase-luciferin containing 2.0 mg lyophilized firefly luciferase-luciferin reagent was injected successively into each sample. The luminescence was determined quantitatively by photon counting in a scintillation spectrometer (Nuclear Chicago, Model 720) with the coincidence circuit made inoperative (Stanley and Williams, 1969). Five successive counting periods of 10 s each were started 10 s after the addition of the reagent. The mean of the last two counts was used as a measure of each sample's luminescence activity (Handa and Bessan, 1980). The photon counting detected by this procedure reflects arbitrary counts, and not absolute number of photons emitted. The cytotoxic effect of antiserum is expressed as light production, measured as cps; or as percentage of cells lysed. The latter was calculated according to the following formula: % cell lysed = (light production of negative control) -- (light production of antiserum-treated sample) X 100 (light production of negative control)
The negative control consisted of treatment with complement and serum from a non-immunized animal. P A - S R B C rosette assays were performed according to Koo and Goldberg (1978) with minor modifications.
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Results
A TP content of rat epididymal sperm With the luciferin-luciferase reagent, light produced was linearly related to the ATP concentration (Fig. 1). Similarly, light produced was linearly proportional to extracts of increasing concentrations of spermatozoa (Fig. 2). According to these results, it was calculated that the ATP content in epididymal spermatozoa is approximately 55 pmol/106 cells. The slope of the standard curve obtained with varying numbers of spermatozoa is steep enough to permit a differentiation of 10-15 % in light production. Effects of altering concentrations of luciferase-luciferin reagent on the sensitivity of the assay To assure a sufficient supply of luciferase-luciferin reagent, we determined the minimal quantity required for a linear, measurable relationship between light production and numbers of untreated spermatozoa (Fig. 3). While 2 mg reagent per sample is adequate for the analysis of the ATP content in spermatozoa (up to 5 X 106), a decrease of the luciferase-luciferin reagent to 0.5 or 1.0 mg markedly reduced the sensitivity (Fig. 3). Effect of complement concentration on A TP content and sperm cytotoxicity With heat-inactivated anti-serum to spermatocytes (1:64), addition of heat-inactivated guinea pig complement (1:4 or higher dilution) did not alter the relative
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A T P content of epididymal spermatozoa samples (Fig. 4). The addition of active complement however, resulted in a nearly linear decrease in A T P content between the dilutions of 1 : 4 and 1 : 32 (Fig. 4). The safe working range of active complement concentration was determined by treating spermatozoa samples in the absence of antiserum. The data indicated that the guinea pig complement had little or no cytolytic effect when used at a dilution of 1 : 16 or greater (Fig. 4). On the basis of these results, we used a dilution of 1 : 20 of complement for all subsequent cytotoxic tests.
Effects of rabbit anti-rat spermatocyte serum on A TP content and sperm cytotoxicity The sensitivity of the sperm cytotoxicity test based on the A T P content of unlysed cells was investigated using rabbit anti-rat spermatocyte (positive control) serum. The titration curve obtained in samples incubated with serial dilutions demonstrated that the cytotoxic titre of this antiserum was between 1 : 128 and 1 : 256 and that a 10-15% difference in relative A T P content is readily measurable (Fig. 5). Titration of H - Y antisera with the luciferase-luciferin procedure for the determination of A TP content in spermatozoa and cytotoxicity Using the experimental conditions established above, we determined the cytotoxicity of different samples of anti-H-Y sera. Sera containing monoclonal antibodies against H - Y (1 : 8), either u n a b s o r b e d or absorbed with female spleen cells,
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322 lysed significantly more spermatozoa than antiserum absorbed with male spleen cells (Fig. 6). We obtained similar results with conventional rat anti-H-Y antiserum (Fig. 7). Conventional mouse anti-H-Y sera were usually found to have lower titres, although there was considerable individual variation with cytotoxicity titres ranging from 1 : 4 to 1 : 32 (data not shown). We assessed levels of cytotoxic activity of these mouse antisera with the A T P assay, and we assessed the same antisera with the P A - S R B C rosette assay. Results from both sets of assays were generally comparable, with excellent correspondence between sera having high activity (Fig. 8). The absence of demonstrable cytotoxicity in sera from mice 12, 13 and 16 correlates well with the low percentage of rosettes formed by sera from the same individuals. The high levels of cytotoxicity of sera from mice 1, 3, 4, 6, 8 and 11 correlate well with the high percentage of rosettes formed by sera from these animals. In sera from remaining mice tested (2, 5, 7, 10, 14 and 15), in which cytotoxicity levels were intermediate judged by the A T P assay, the percentage rosette formation was variable (Fig. 8).
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Discussion
For the assessment of levels of H-Y antigen on sperm, conventional cytotoxicity tests of sperm based on uptake of supravital dyes in lysed cells are notoriously difficult to score objectively. We have tried trypan blue, eosin red and fluorescent dyes, but we have not been satisfied that entry of any of these into sperm provides a reliable, objective measure of sperm cytotoxicity. In other attempts to assess H-Y antisera, we have measured the uptake of radioactive amino acids by cultured male somatic cells previously subjected to H-Y antisera and complement. We have also searched for evidence of specific binding of 125I-labeled protein A to sperm or cultured somatic cells, dependent on prior incubation of these cells with H-Y antibodies. Neither of these sets of procedures provided quantitative assays for H-Y antigen (unpublished observations).
324
The PA-SRBC rosette assay described by Koo and Goldberg (1978) offers a less subjective method for H-Y antisera titration. It is, however, difficult to distinguish between erythrocytes which are truly bound to the sperm and those which superficially adhere or partially superimpose upon the sperm. The firefly luciferase-luciferin method is generally acknowledged as a highly specific, sensitive, simple and reproducible method for the quantitative determination of ATP. Results presented above demonstrate that this technique can be readily adapted for the detection of male-specific antigen(s), and for the titration of H-Y antibodies from various sources. Procedures utilized obviate the need for tedious counting procedures. A quantitative analysis of ATP levels in sperm provides an accurate measure of cell lysis, thereby replacing the need to identify lysed cells microscopically, or to count RBC-sperm rosettes when assessing the potency of antibodies directed against antigens on the surfaces of spermatozoa. Replacement of these subjective aspects by objective analysis of ATP permits a more reliable estimate of the cytotoxic potency of various antisera. For these reasons, we place more weight on the ATP assay than on the PA-SRBC rosette assay in evaluating the sera from individual mice reported in experiments summarized in Fig. 8. Since the ATP content of a given cell type is relatively constant, good precision and reproducibility are possible.
Acknowledgments We express our appreciation to Dr. Maria Zenzes for her useful comments. The excellent technical assistance of Edna Cartwright and Lily Chen is gratefully acknowledged. This work was supported by grants from the N I H (grant 5R01HD 11741-02) and MRC of Canada (MT 3292).
References Goldberg, E.H., Boyse, E.A., Bennett, D., Scheid, M. and Carswell, E.A. (1971) Serological demonstration of H-Y (male) antigen on mouse sperm. Nature (London) 232, 478-480. Handa, A.K. and Bressan, R.A. (1980) Assay of adenosine 3',5'-cyclic monophosphate by stimulation of protein kinase: a method not involving radioactivity. Anal. Biochem. 102, 332-339. Koo, G.C. and Goldberg, C.L. (1978) A simplified technique for H-Y typing. J. Immunol. Methods 23, 197-201. Ohno, S. (1979) Major Sex Determining Genes. Springer-Verlag, Switzerland. Stanley, P.E. and Williams, S.G. (1969) Use of liquid scintillation spectrometer for determining adenosine triphosphate by the luciferase enzyme. Anal. Biochem. 29, 381-392. Suominen, J.J.O., Multamaki, S. and Djupsund, B.M. (1980) A new method for measurement of cytotoxic antibodies to human spermatozoa. Arch. Androl. 4, 257-263. Tung, P.S. and Fritz, I.B. (1978) Specific surface antigens on rat pachytene spermatocytes and successive classes of germinal cells. Dev. Biol. 64, 297-315. Webster, J.J., Cheng, J.C., Manley, E.R., Spirey, H.L. and Leach, F.R. (1980) Buffer effects on ATP analysis by firefly luciferase. Anal. Biochem. 106, 7-11.