BIOLOGY

OF REPRODUCFION

41, 533-542

(1989)

Cell-Mediated Immune Responses to Sperm Antigens: Effects on Mouse Sperm and Embryos1 RAJESH Department Albert

K. NAZ2’3

and KAPIL

MEHTA4

of Obstetrics and Gynecology3 Einstein College of Medicine Bronx,

Department M. D. Anderson

New

York 10461

and of Clinical

immunology4

Hospital

Houston,

and Tumor

Texas

institute

77030

ABSTRACT Sperm

antigens were assessed for their ability to induce cell-mediated immune (CMI) responses. Purified antigen (FA-I), protamine, and the lithium diiodosalicylate (LJS)-solubilized sperm preparation activated presensitized lymphocytes to secrete soluble mediators that activated macrophages and significantly inhibited sperm motility and embryonic development. The FA-), however, was the most potent antigen in inducing proliferative response as well as the release of soluble mediators. US-sperm preparation, which contained numerous antigens, showed the least activity. The unsensitized control spleen cells did not secrete any factor(s) when activated with the antigen. In conclusion, these results indicate that sperm antigens can specifically induce (CMI) factors that have detrimental effects on sperm motility and pre#{252}nplantation embryos. These findings may have potential clinical implications for humans, especially in immwwlogic and unexplained infertility, recurrent abortions, and development of antisperm contraceptive vaccines.

fertilization

INTRODUCTION

used as an immunogen. Therefore, further studies to understand the immune mechanisms responsible for suppression of fertility require the use of defined antigens. Recently, we isolated and characterized a spermspecific glycoprotein, the fertilization antigen (FA-1), from human and mouse male germ cell plasma membranes (Naz et a!., 1986b). The tissue-specific but species-cross-reactive monoclonal antibodies against FA-1 inhibit human sperm penetration of zona-free hamster ova and block penetration of mouse sperm into mouse oocytes (Naz et a!., 1984). The FA-1 causes a reduction of fertility in actively immunized female rabbits by mechanisms involving an inhibition of fertilization (Naz, 1987a). Interestingly, FA-1 is also involved in involuntary immuno-infertility in humans (Naz, 1987b). The cell-mediated immune (CMI) responses are readily induced by foreign cellular and viral antigens (Henry, 1970; Zinkernagel and Doherty, 1974). However, CMI responses to sperm antigens that may be relevant to infertility have remained virtually unexplored. The objective of the present studies was to investigate whether the well-defined sperm antigens could activate presensitized lymphocytes to secrete the soluble products (lymphokines), and whether these soluble products

Antispermatozoal antibodies have been implicated as cause of infertility in humans (reviews by Menge, 1980; Bronson et a!., 1984). Immunization of male and female animals of various species with extracts of sperm or mature testes results in a significant inhibition of fertility by causing fertilization failure (Katsh, 1959; Edwards, 1964) and/or by causing postfertilization preimplantation embryonic mortality (Naz et al., 1983; Menge and Naz, 1988). These antifertility effects have been attributed to antispermatozoal antibodies developed after immunization. In these studies, whole sperm or mature testes containing numerous antigens were

a

Accepted May 5. 1989. Received September 30, 1988. 1This work was supported by grants from NIH (HD-24425 to R.K.N) and from the Program for Applied Research on Fertility Regulation (PARFR) under a cooperative agreement with the United Stales Agency for Internstional Development (MD) (DOE-0546-A-1003-00 to R.K.N) and a grant from NIH (CA-38751 to K.M). The financial assistance provided through INDO-USAID (to R.K.N) is gratefully acknowledged. This work was presented at the 36th Annual Meeting of the Society for Gynecological lnves*igatOflA San Diego, CA, March, 1989. ‘Reprint requests: Dr. Rajesh K. Naz, Reproductive Immunology Research Laboratory, Ullmann Research Building #123, Albert Einstein College of Medicine, Bronx, NY 10461.

533

NAZ

534

AND

could affect sperm motility and embryonic development. These studies also explored, in general, the CMI responses against sperm and their potential relevance to involuntary infertility, recurrent abortions, and development of antisperm contraceptive vaccines. MATERIALS

AND

METHODS

MEHTA et a!., 1986b). Briefly, the washed murine epididymal sperm were solubilized in 0.3 M LIS in 0.05 M IrisHC1 (pH 8.0) containing 1 mM phenylmethylsulfonyl fluoride and 5 mM soybean inhibitor at room temperature for 30 mm, at 4C for 2 h, and then centrifuged at 25,000 x g for 30 miii. The LIS-solubiized sperm supematant was dialyzed against 0.15 M LiC1 and then against 0.05 M Tris-HC1 (pH 8.0).

Antigens immunization

Three different sperm antigen preparations were used for immunization, these were FA-l (a surface antigen), protamine (an intrinsic nuclear antigen), and lithium diiodosalicylate (LIS)-solubiized mouse sperm preparation containing numerous antigens. FA-1. FA-l was purified from LIS-solubilized mouse testes by immunoaffmity chromatography involving monoclonal antibodies that inhibit fertilization (Naz et a!., 1984, 1986b). Two-dimensional polyacrylamide gel electrophoresis (2-D-PAGE), high-performance liquid chromatography (HPLC), density gradient ultracentrifugation, and amino acid analysis indicated that the purified antigenic molecule is composed of a monomer of 23,000 M1 and/or a dimer of 47,000-50,000 M (Naz et a!., 1986b). Each batch of FA-1 was tested for its homogeneity. Only those batches that showed specific bands of 23,000 and/or 47,000-50,000 M in sodium dodecyl sulfate (SDS)-PAGE when stained with ultrasensitive silver stain were used in the present study. SDS-PAGE was performed according to the method of Laemmli et a!. (1970). In SDS-PAGE, the antigen mostly showed its dimeric form. The antigen solution was extensively dialyzed against 0.05 M tris(hydroxymethyl)aminomethane (Tris)-HC1 buffer (pH 8.0) before use. Protamine. Salmon protamine, which is commercially available (salmine, free base, Grade VI, Sigma Co., St. Louis, MO) was used for immunization. Protamines are small (-i-4 were pooled (from 2-3 animals) and used in the present experiments. Ham’s F-b medium was prepared as described elsewhere (Naz et a!., 1986b). Each liter of Ham’s F-10 medium (pH 7.58-7.69, 280 Osm/kg) contained Ham’s F-10 powder (GIBCO), 75 mg penicillin 0, 75 mg streptomycin, 2.106 g of sodium bicarbonate (NaHCO3), 1 mmol of calcium lactate, and 0.5 mmol of potassium bicarbonate (KHCO3) dissolved in HPLCgrade water (Chrom HPLC Mallinckrodt, Paris, KY). Supernatants (50 p1) generated from antigen-challenged or mitogen-challenged spleen cells were mixed with Ham’s F-i0 medium (50 p.1) containing 5-8 x 106 motile sperm/mi placed in wells of microwell Terasaki ELISA plates (Falcon, Oxnard, CA). Antisera collected from mice immunized against FA-1, protamine, or LISsperm extracts were also tested (1:10 dilution) for their

NAZ

536

AND

MEHTA Assay for embryotoxic activity. The 2-cell mouse embryo bioassay was performed as described elsewhere (Naz et a!., 1986b). Briefly, virgin female mice, 8-15 wk old, of C57 BL/6JXCBAJJ or CD-i (Charles River, Kingston, ON) were used for the study. Ovulation was induced by i.p. injection of 7.5 U of human menopausal gonadotropin (Pergonal, Serono Labs, Inc., Randolph, MA) or 7 U of pregnant mare’s serum (Sigma Chemical Co.). After 48 h, the mice were injected with human chorionic gonadotropin (hCG, Sigma Chemical Co.) and kept overnight for mating with male mice of proven fertility (2 or 3 females with i male per cage). The next morning, the female mice were checked for vaginal plug formation. The mated females were killed 36 h after hCG injection and their oviducts were flushed with Dulbecco’s phosphate-buffered saline (pH 7.4, 280 mOsm/kg, GIBCO). The 2-cell embryos recovered were washed with Dulbecco’s phosphate-buffered saline and incubated (37#{176}C, 5% C02:95% air mixture) in the pre-equilibrated culture medium for 72 h. The percentage of 2-cell embryos that developed into blastocysts was recorded as the blastulation rate. Embryos (10-15) were cultured in 100-pI drops of Ham’s F-!0 medium containing 50% culture supernatant (v/v). Each sample was tested at least 3 times with embryos from different mice. Only those samples that were positive for the macrophage-activating factor and also demon-

effects

on sperm motility. The percent and progressive was recorded at the start and at various time intervals up to 2 h after incubation, as described elsewhere (Naz and Talwar, 1986). The progressive motility was graded from 0 to 5, 0 indicating no sperm movement; +1 indicating movement without forward progression; and +3, +4, and +5 indicating sperm movement with increasing degree of forward progression from slight to most vigorous (Naz and Taiwar, 1986). Samples were run in duplicates and incubated at 37#{176}C in a humidified 5% C02: 95% air incubator. Each sample was tested at least 3 times using different sperm samples. The supernatants from unstimulated cell cultures and those that were negative for the macrophage activating factor served as controls. For immunoabsorption studies, the supernatants (50 p.1) from spleen cells sensitized in vivo and activated in vitro with FA-1 were incubated overnight at 4#{176}C with rabbit monospecific polyclonal antibodies (10 p.1) against recombinant mouse y-interferon (Genentech Inc., South San Francisco, CA) or with monoclonal antibodies against tumor necrosis factor (TNF, Genentech Inc.). The absorbed supernatants were then mixed with Ham’s F-I0 medium (40 p1) containing 5-8 x 106 motile sperm/mi, and the percent motility and progressive motility were recorded as above. The supernatants incubated overnight without antibodies and the antibodies alone served as controls. motility

lOSgel M.W.x104

MW.x105

- 97 -68

0.20 E C 0

0.15

cJ

0.10

I

bc

0.05

2.8

0.0 2

6

8

10

12

14

16

Fraction

18

20

22

24

26

No.

FIG. I. The FA-I was purified from lithium diiodosalicylate (US)-solubilized mouse testes using immunoaffinity chromatography (Naz at *1., 1986b). The mouse testes were freed of tunica albuginea and dissolved in 0.3 M LIS in 0.05 M Ths-}ICI buffer (pH 8.0) containing 1 mM phenylmethylsulfonyl fluoride and 5 mM soybean trypsin inhibitor. The solution was passed through an immunosorbent column prepared by coupling IgG (isolated from anu-FA-1 monoclonal antibody ascites) to Sepharose-4B activated by CNBr. The column was washed with buffer (pH 8). and the bound proteins were subsequently eluted with pH 4(t) and then with pH 2.8(1) glycine-HCI buffer. The fraction eluted at pH 2.8 showed a single band of 47,000-50,000 Mr (dimer) (Lane b, i,iset) or a single band of 23,000 Mr (monomer) (Lane c, inset) in SDS-PAGE. In SDS-PAGE, FA-1 mostly showed the band corresponding to its dimeric form. LIS-solubilized sperm preparation demonstrated numerous bands (Lane a, tact). Protamine, when run in 15% acid-urea gel, demonstrated two bands between 6000-8000 Mr (Lane d, Inset). The gels were calibrated by using a mixture of protein standards of known molecular weights.

SPERM TABLE

1. Effects

of sperm antigens

on lymphocyte

proliferation Serum

Activating agent

Source of lymphocytes

Mitogen PHA PHA PHA PHA PHA Sperm antigens FA-l FA-I FA-I Protamine US-sperm

ANTIGEN-INDUCED and release

CM1

of macrophage-acuvating

antibody

537 factor

(MAF)

Proliferative MAP activity

responset

‘#{176}

nrfr’

nrht

Buffer alone (control) Buffer + FCA-ireated FA-1-sensitized Protamine-sensilized US-sperm-sensitized

0/5 0/5 8/10 4/5

5/5

3/5

5/5 5/5

Buffer alone (control) Buffer + FCA-trcated FA-l-sensitized Protamine-sensitized LIS-sperm-sensitized

0/5 0/5 8/10 4/5

0/5 0/5 7/10 4/5

3/5

3/5

/0C

SI 85.00 114.00 120.00 45.00 42.00

5/5

9/10

0.98 1.20 26.00 1800 12.00

± ± ± ± ±

2300d 3O.OO 16.00e 12#{149}00e 8.OO

±

O.3O

± ± ± ±

0.20 8.0051 7.0052 4.00

5Number of animals whose sera had antibody ELISA titers of >1:20 against respective antigen vs. total number bExpressed as the mean stimulation index (SI) ± SD, obtained by dividing the CPM of the mitogen-stimulated cNum

of animals

whose

spleen

cells gave significant

reaction

(>1 SI or >20% cytotoxicity)

1evs.respectivecontrol(d),p=0.01_0.001;g1-g6vs.respectivecontrol(f),p=0.01_O.001; vs. g6,p = 0.01; g2 vs. g3, insignificant. All others were insignificant

T-interferon or TNF, as described above, were mixed with 40 p.1 Ham’s F-10 medium and investigated for embryotoxic effects. Supematants incubated overnight without antibodies served as controls. Analysis

Significance dent’s i-test.

of differences

was

analyzed

vs. total number

Cytotoxicity 52 96

5/5

9/10

by

Stu-

RESULTS

The immunoaffinity-purified FA-1 showed a single band of 23,000 Mr (monomer) and/or 47,000-50,000 Mr (dimer) in SDS-PAGE (Fig. 1, Lane c and Lane b, inset, respectively), and LIS-sperm preparation demonstrated numerous bands of various molecular identities (Fig. 1, Lane a, inset). Protamine showed a broad band comprising 2 peptides between 6000 and 8000 Mr in acid-urea gel electrophoresis after staining with silver nitrate (Fig. 1, Lane d, inset). The purified antigen (FA-l) or protamine or partially purified (LIS-sperm) antigens were tested for their ability to trigger the CMI response against sperm and/or embryonic development. Groups of mice were immu-

± ±

(%)

6d 3C

82 ± 8e 72 ± 15C 69 ± lO

3/5

10 ±

0/5 1/5 8/10

16 ± 72 ±

4/5 3/5

49 ± 31 ± 1256

7

immunized; nr = number responding. cultures by the CPM of the control cultures. tested;

or

=

number

responding.

g2andg3vs.gl,p=0.01-0.OOl;g5andg6vs.g4,p=0.001; vs. respective

strated sperrnicidal activity were investigated for embryotoxic activity. The supernatants from unsthnulated cell culture and those that were negative for the macrophage-activating factor, as well as for spermicidal activity, served as controls. The supematants (50 p.1), immunoabsorbed overnight with antibodies (10 jil) against

Statistical

activity.

g5

controls.

nized with antigens; subsequently, their spleen cells were stimulated in vitro with specific (antigens) or nonspecific mitogens (PHA). Table I demonstrates that all 3 sperm-antigen preparations were able to stimulate the spleen lymphocytes from sensitized animals, although FA-1 induced a much higher response than did LIS-sperm or protamine antigen. In general, animals showing higher antibody ELISA titers in serum showed higher proliferative response and release of soluble mediators on in vitro activation with specific antigens. Spleen cells from all the animals responded equally well to the nonspecific mitogen (PHA). except those from protamineor US-sperm-sensitized animals, which showed moderate, but consistent, low response to PHA. We next looked at the production of soluble mediators by spleen lymphocytes that had been activated with specific or nonspecific mitogens. Supernatants from spleen-cell cultures were examined for their ability to prime the mouse peritoneal macrophages in an UPStriggered activation assay to inhibit mouse adenocarcinoma (EMT-6) cell growth. As shown in Table 1, there was a good correlation between the proliferative response and induction of tumor cell-killing activity in PHA-stimulated or antigen-stimulated spleen cell cultures. Supernatants from spleen cells activated by PHA or FA-1 or protamine induced a significantly stronger macrophage-mediated anti-tumor activity against EMT6 target cells, compared to unstimulated or LIS-spermactivated cultures. The spleen cells from control mice failed to proliferate or release macrophage-activating

NAZ

538 TABLE

2. Effects

of conditioned

supernatanis

on sperm

Percent (mean

Source of lymphocytes

Supernatants absorbed with:

0

None (control) FA- 1 FA-1 FA-1 FA-1 Protamine LIS-sperm PHA

Buffer + FCA-treated Buffer + FCA-trcatcd FA-l-sensitized FA-1-sensitized FA-1-sensitized Protamine-sensitized LIS-sperm-sensitized Untreated

None None None Anti-yintcrferon Anti-TNF None None None

92 92 92 92 92 92 92 92

and at vs. respective

insignificant

vs. respective

control.p=0.01-0.05;

± ± ± ± ± ± ± ±

5 5 5 5 5 5

5 5

bl andb2 vs. respective

3. Effects

motility ± SD) mm

2 h

92 92 73 82 88 77

5 5

89 ± 89 ±

± ±

control,p=0.004-0.006;

6 6

± 31b ± 20’

± 28 ± 32’ ± 12b ± 29’ at andb2

30-45

mm

2 h

4.5 4.5

± 0.4 ± 0.4

4.5 ± 0.3 4.5 ± 0.4

4.3 45

4.5 4.5 4.5 4.5 4.5 4.5

± ± ± ± ± ±

3.7 ± 3.9 ± 4.2 ± 3.7 ± 4.2 ± 3.8 ±

1.0 1.0 0.8

1.9 ± 2.9 ± l.4hi 2.1 ± 1.5

1.0

2.2 ±

0.3 0.9

3.1 ± 06b2 2.3 ±

0.4 0.4

0.4 0.4 0.4 0.4

vs. bl,p=0.001;

b2 vs. al,p=0.001.

± 0.3 ± 0.3

1.111

Others were

of conditioned

supernatants

on 2-cell

embryonic

progressively up to 2 h. Both percent and progressive motility showed similar effects; with a decrease in percent motility, there was a concomitant decrease in progressive motility. The supernatants absorbed with antibodies against y-interferon or TNF showed less deleterious effects on sperm motility than the corresponding unabsorbed supernalants. The effects of absorption with antibodies against TNF were less drastic than those of absorption with 1-interferon and were statistically insignificant. The murine anti-FA-1 antisera collected from various (n = 3) mice having ELISA titers of >1:512 did not show any effect on sperm motility (both percent as well as progressive) over 2 h of observation. Similarly, murine anti-protamine antisera (n = 2) and anti-LIS-sperm antisera (n = 2), having ELISA titers of 1:2056 and >1:512 against protamine and LIS-sperm extract, respectively, did not reduce motility. Supernatants from activated spleen cells also inhibited 2-cell embryonic growth. Supernatants from leukocytes activated with PHA caused a significant

growth.

Source of lymphocytes

Supernatants

2-Cell

absorbed

embryos

None (control)

Buffer

FA-l FA- 1 FA-1

FA-1-sensitized FA.l-sensitized FA-l-sensitized

Protamine LIS-sperm PHA

Prolamine-sensitized LIS-sperm-sensitized Untreated

None None Anti-y interferon Anti-TNF None None None

controls.

38 69 41 44 62 50

0

± SD)

controls.

Activating agent

respective

motility

Progressive (mean

30-45

± 18’ ± 17’ ± 5 ± 23 87 ± 8 77 ± 20

factor (y-interferon) activity following their exposure to FA-1. None of the supematants showed any detectable level of antibodies against any antigen preparation. Supernatants that were positive for macrophage-activating factor also showed a strong spermicidal activity. Supernatants from splenocytes stimulated with PHA caused a significant decrease in percent motility as well as progressive sperm motility over 2 h of observation, compared to controls (Table 2). Supernatants from in vivo-sensitized and, subsequently, in vitro-activated spleen cells with FA-1 showed a drastic reduction in percent motility as well as progressive motility (Table 2). Similar results were obtained with supernatants from protamine-sensitized spleen cells activated with protamine. Supernatants from LIS-sperm-sensitized spleen cells activated with US-sperm preparation also induced a decrease in sperm motility (percent as well as progressive), though the effects were not as drastic as the supematants from FA-l-activated or protamine-activated spleen cells (Table 2). The effect was observed as early as after 30 mm of incubation, and it increased

TABLE

MEHTA

motility.

Activating agent

a,ba

AND

+

FCA-treated

with:

Embryos developed to blastocysts

Blastulation rates (mean ± SD) (%)

128 72 71

132 27 26 18

86

30

86

63 32

88 22 36 25 35 3 36

150

89

cultured

± ± ± ± ± ± ±

10 2111 10 1712

l2 15’

SPERM

FIG. 2. Photoinicrographs of mouse embryos. Two-cell stimulated (c), or US-sperm-stimulated (d) leukocyte culture blastocysts (e) within 72 h. Note the presence of degenerated from US-sperm-stimulated leukocytes. (x 300.)

ANTIGEN-INDUCED

CM1

539

mouse embryos (a) cultured in the presence of PHA-stimulated (b), FA-1-swnulaied or prolaminesupernazants. Two-cell murine embryos cultured in the presence of medium control developed to form embryos arrow lying side by side with btastocysis (d) in the culture medium containing supernatants

(p80% mice. Mice that did not reveal activity demonstrated low (70% cross-reaction with murine TNF. The effects may have been more significant with polyclonal antibodies against murine TNF. It is equally possible that 1-interferon may be one of the major bioactive factor(s) related to the reproductive process, as it may be secreted in larger quantity than TNF on activation with sperm antigens in in vitro. The antibodies alone did not show any direct effect on sperm motility, indicating that the effects were mediated through neutralization of the factors secreted in the supematants from spleen cells. It appears that several factors are secreted in the supematants that are cytotoxic to sperm or embryos. In conclusion, our data indicate that sperm antigens can specifically activate presensitized lymphocytes to secrete soluble factors that can adversely affect sperm motility and embryonic development. If demonstrated in vivo, the findings of this study will have potential clinical implications in humans, especially in immunologic and unexplained infertility, recurrent abortions, and development of antisperm contraceptive vaccine. Both male and female reproductive tracts are immunologically dynamic systems. Immune reactive cells, including lymphocytes and macrophages, reside ubiquitously in the genital tracts of both sexes (Haney et al., 1983; Olsen and Shields, 1984). Sensitization of immune cells with sperm antigens can induce both humoral and cellular immune responses affecting the reproductive processes at prefertilization as well as at postlertilization stages. Based upon these results, the deliberate induction of CMI responses by immunization with FA-1 could enhance the effectiveness of the antisperm contraceptive vaccine (Naz, 1988).

CM1

541 ACKNOWLEDGMENTS

Mr)

We thank Iris Barad,

Janice

Bedard,

and Elaine

Glikson

for typing

this

manuscript

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CS. 19700. A cytolytic system for the in vitro detection of cell-mediated immunity to soluble antigens. I Immunol 105:919-27 Hill JA, Florina H, Anderson DJ, 1987. Products of activated lymphocytes and macrophages inhibit mouse embryo development in vitro. I Immunol 139: 1150-2254 Katsh S. 1959. Infertility in female guinea pigs induced by injection of homologous sperm. Am J Obstet Gynecol 78:276-78 Laenunli UK, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Land) 220:680-85 Lal RB, Lynch Ti, Nulman TB, 1987. Bnsgw malayl antigens associated with lymphocyte activation in filariasis. 3 Immunol 139:1652-57 McShane PM. Schiff I, Trentham DE, 1985. Cellular immunity to sperm in infertile women. JAmMed Assoc 253:3555-58 Mebta K, aaringbuld P. Lopez-BeresteinG, 1986. Ampholericin B inhibits the serum-induced expression tissue transglutmiiin in murine pcritcneal macrophage. 3 Immunol 136:4206-12 Mehta K, Claringbold P. Lopez-Bernstein G, 1987. Suppression of macrophage cytostatic activation by serum retinoids: a possible role for transglutmin. I Immunol 138:3902-06 Menge AC, 1980. CliniCal immunological infertili . diagnostic measures, incidence of antisperm antibodies, fertility and mehnimc. Dhlndsa DS. Schumacher GB (eds.), New York: Elsevier-North Holland, pp. 205-25 Menge AC, Naz RK, 1988. linmunologic reactions involving sperm cells and preiinplantation embryos. Am I Reprod Imnumol Microbiol 18:17-20 Nez RK, 1987a. The fertilization antigen (PA-i) causes a reduction of fertility in actively immunized female rabbits. J Reprod Immunol 11:117-33 Nez RK, 198Th. Involvement of fertilization antigen (PA-I) in involuntaiy immunoaffinity in humans. J Clin Invest 80:1375-83 Nez RX, 1988. The fertilization antigen: applications in immunocontraception and infertility in humans. Am J keprod Immunol Microbiol 16:21-27 Nez RK, Alexander NJ, Isahakia M, Hamilton MS, 1984. Monoclonal antibodies to a human cell membrane glycoprolein that inhibits fertilization. Science 225:342-44 Nez RX, Deutsch 3, Phillips TM, Menge AC. Fisch H, 1989. Sperm antibodies in vasectoinized men and their effects cc fertilization. Biol Reprod 40: 163-73 NazRK, Janousek I, Moody T, StillmanRJ, 1986s. Factors influencing cnibtyo bioassay: effects of proteins, aging of medium and surgical glove coatings. Fertil Steril 46:914-19 Nez RK, Phillips TM, Rosenbium BB, 1986b. The characterization of the fertilization antigen I for the development of contraceptive vaccine. Proc Nati Acad Sci USA. 83 :5713-17 Nez RK, Saxe JM, Menge AC. 1983. Inhibition of fertility in rabbits by monodecal antibodies against sperm. BlOt Reprod 28:249-54 Nez RK, Talwar GP, 1986. Reversibility of azoospermia induced by bacillus calmeue-Guerin, 3 Androl 7:264-69 Olsen GP, Shields 1W, 1984. Seminal lymphocytes, planna and AIDS. Nature (Land) 309: 116-17 Otresen EA, Weller PF, Heck L, 1977. Specific cellular unrcaponsivmess in human filariasis. Immunology 33 :413-21

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MEHTA J Immunol Methods 1:273-87 Tsujcmolo M. Yip YK. Vilcek 1, 1985. Interferon-y enhances expression of cellular receptors for tumor necrosis factor. Proc Nail Acad Sci USA 82: 7626-30 Tucker SB. Pierre 1W, Jordan RE, 1977. Rapid identification of monocytes in mixed mononuclear cell preparation. J Inununol Methods 14:267-69 Voller A, Bidwdll D, Bartlett A, 1976. Microplate enzyme immunoassays for the iinmunodiagnosis of vinis infections. In: Rose NR. Fredman H (eds.), Manual of Clinical Immunology. Washington, DC: American Society for Microbiology, pp.506-12 Zinkernagel RM. Doherty PC. 1974. Immunological surveillance against a!tered self components by sensitized T-lymphocytes in lymphocytic choriomeningitis. Nature (Land) 251:547-48