Radioisotope assay of sperm-binding globulin

RADIOISOTOPE

ASSAY OF SPERM-BINDING

R. W. Noyes,

M. D.,

and M. R.

University

C.

B.

Camyre,

GLOBULIN

Odom, M. D. M.

S.

Departments of Anatomy and Obstetrics-Gynecology of Hawaii School of Medicine, Honolulu, Hawaii,

96822

ABSTRACT lniectinq doe rabbits with 6 X lo7 rabbit or human spermatozoa plus ad]uvant-did not increase sperm-binding globulin leveis in their serum, nor was their fertility significantly altered. The radioisotope assay results done in our laboratory corresponded closely with results of hemagglutination titers, sperm macroagglutination and immobilization tests done in another laboratory on samples of the same immune and control globulins. Whether these various tests measure the same or different components in globulin, and which components should be referred to as “antibodies” to sperm proteins, remains to be elucidated.

Supported in part by grant HD-02066 of the NIH. Dr. Odom was a Postdoctoral Fellow of the East West Population Institute, and of the Ford Foundation’s program for reproductive physiology, University of Hawaii.

Acknowledgments: We are grateful to A. C. Menge, Ph.D., Department of Obstetrics and Gynecology, University of Michigan School of Medicine, for inspiring this work and for sending us immune globulin. Accepted for Publication November 10, 1972

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LNTRODUCTION The potential usefulness of immunization of women against spermatozoa as a contraceptive technique depends on how effectively and how safely immunization might be carried out. When animals are immunized with large doses of seminal proteins and Freund’s adjuvant, skin sloughs and severe systemic reactions often result. Menge (1) immunized doe rabbits with rabbit epididymal sperm or whole semen, using polyribonucleotides as adjuvant, and found that serum antisperm antibodies could be detected by passive hemagglutination, sperm immobilization and sperm agglutination tests. Fertilization, implantation, sperm transport and sperm motility were inhibited. Skin and systemic reactions were much less with polyribonucleotide adjuvant than with Freund’s adjuvant. The present experiment5 had 3 objectives: first, to determine how many doe rabbits might develop sperm-binding globulins after being injected with about the same number of spermatozoa as normally enter their to compare the effectiveness of upper reproductive tracts; second, Freund’s adjuvant with polyribonucleotide adjuvant; and third, to compare sperm agglutination and sperm immobilization techniques with a previously reported radioisotope tagging method for sperm-binding globulins. MATERIALS

AND METHODS

Semen was collected from Buck 44 by means of an artificial vagina and stored at -40°C. The frozen ejaculates were thawed, pooled, centrifuged, and the button containing spermatozoa and seminal solids was suspended in 0.85% saline at a concentration of 5 X 107 sperm per ml. Some of this was later diluted and used for injection and some was used as the test antigen in the radioisotope assay. Further semen was collected from 8 different bucks and from a human donor, and these specimens were treated the same as Buck 44’5 semen. Doe rabbits of mixed breeds were divided into 6 treatment groups and injected as follows: I) Sperm from Buck 44 plus Freund ‘s; 2) Sperm from Buck 44 plus polyribonucleotides; 3) Saline plus Freund’s; 4) Saline plus polyribonucleotides; 5) Human sperm plus Freund’s; and 6) Human sperm plus polyribonucleotides. In treatme t groups I, 2, 5 and 6 the number the adjuvant of sperm injected was kept at 10 X 10 g in 0.2 ml sal ine, was 0.2 ml., and when emulsified together,the injection volume was 0.4 ml. Freund’s complete adjuvant (Difco), and salts of polyadenyl ic and polyridylic acids (Calbiochem) 0.2 mg /ml solutior$were stored at -40°C. The suspensions were injected into subscapular and gluteal muscles at weekly intervals for 6 weeks. In addition to the six treatment groups already two globulins prepared by Dr. A. C. Menge and mailed as unknowns. Group 7 was lyophylized globulin from

58

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described, we tested to our laboratory a rabbit injected

1973

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CONTRACEPTION

with rabbit seminal lyophylized globulin and polyribonucleotides.

plasma and polyribonucleotides, from each of 3 rabbits injected

and group 8 with rabbit

was sperm

Blood was drawn from ear vessels at 0, 2, 4, 6 and 8 weeks of treatment, was clotted, and the serum globulins were three times precipitated with 14, 12, and 12 gms percent of sodium sulfate. The final precipitate was dissolved in distilled water, lyophylited without prior dialysis, and stored at -4°C. The lyophylized globulin was reconstituted in phosphatesaline buffer at pH 7.8 and labelled with ‘3llodine by the method of Greenwood _+ et al After recovery from the Sephadex G-25 column (2) * the concentratron was adjusted to 2 mg protein per ml , and the specific radioactivity was determined. One-half ml of labelled globulin was mixed with 5 X 106 sperm in 0.1 ml of saline in a bovine-serum-albuminpretreated glass test tube for 30 minutes at room temperature. The spermatozoa were then washed three times with distilled water and centrifuged at 1500 G for 20 minutes. The final concentration of the supernatant globulin was l/500 that of the labelled globulin present in the original spermatozoa-globulin mixture. The radioactivity of supernatant plus spermatozoa button was determ’ned and the counts per minute were conEach radioisotope assay verted to mg globulin per 10 8 spermatozoa. was monitored by simultaneously labelling experimental globulin and control globulin derived from pooled buck rabbit’s blood. Each of these globulins was reacted with both specific test antigen (sperm from Buck 44) and control test antigen (sperm from several bucks pooled). The four a) experimental doe’s globulin plus Buck 44’s tests run were therefore: sperm (in 2 cases human sperm); b) experimental doe’s globulin plus pooled bucks (or human) sperm; c) pooled buck’s globulin plus Buck 44’s sperm; and d) pooled bucks globulin plus pooled bucks sperm. Three replicate tubes were run on each of the 4 tests a, b, 4, and II, and the average was reported. The sperm macroscopic agglutination test (3) was performed with final concentrations of 10 X lo6 highly motile spermatozoa and 2.5% gelatin in 0.6 ml saline solution incubated for 2 hours at 37°C. Every experimental test was controlled by means of simultaneously performed dilutions of globulin from uninjected animals as well as globulin known to contain agglutinating antibodies. Fertility was tes ed by inseminating each doe 2 weeks after the last injection with 75 X IO & sperm and injecting 40 I.U. of HCG intravenously Laparotomy was performed 19 days immediately after the insemination. later and the fetuses and corpora lutea were counted. RESULTS Table I gives values for experimental globulin tested against In treatment groups 1 and 2, none Buck 44’s spermatozoa, i.e. test a. of the mean values after sperm inTection were significantly higher than either the pretreatment values or the average value for test 4 (the

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m

z.

\o

0

0

0

+I

+I

0

-f . _

-. +I PI

.

7

Ln N 0

+I

s:

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buck’s globulin plus Buck 44’s average value of 36 assays of Poole El sperm). sperm was 1.6 + 0.7 mg globulin/IO Before the results for the 4 animals in treatment groups I and 2 were averaged, an occasional individual test value was noted to be statistically significantly elevated, and when there were only 2 animals in a group, a few averaged values were also elevated. No biological significance can be attributed to these sporadic statistical elevations. Heterologous sperm injections (treatment groups 5 and 6) did not result in elevated globulin values with either human or rabbit test sperm. Table II, column 1,gives values for experimental globulin tested against pooled buck’s (or human) spermatozoa, i.e. test b. Except for or individual values are significantly group 8, none of the average elevated. When compared with corresponding values fo test < in column 2, s sperm. Treatment the mean of the 8 tests was 2.1 + 0.3 mg. globulin/l0 groups I, 2, 5 and 6, injected w%h sperm, did not have higher values than groups 3, 4 and 7, injected with saline and adjuvant (control) or seminal plasma and adjuvant. Similarly, no group except 8 had positive Lyophyl ized sperm agglutinating tests or occult infertility problems. globulin samples sent to us as unknowns by Dr. Menge correlated well with tests performed in his own laboratory, i.e. radioisotope assay, sperm agglutination, and fertility were normal in group 7 and significantly altered in group 8. he treated Table III gives data obtained by Dr. Menge from 5 rabbits with various antigens, plus radioisotope assay data we obtained from globulin samples sent to Hawaii of the same 5 rabbits. Correlation is fair between the radioisotope assay test and the hemagglutination titer, and is good between the radioisotope assay and the Kibrick test, sperm immobilization titer, and fertility of the treated does. DISCUSSION Spermatozoa are difficult to handle as radioisotope assay test antigens. A large amount of “non-specific” globulin binds to the sperm This results in high control values and and does not wash off easily. relatively small differences between control and experimental values. An example is seen in Table II, treatment group 8, in which 4.3 + 0.83 in an experimental group is only double the control value, mg of globulin though significant at the 0.01 level. Efforts to pretreat the 1.9 mg low-antibody-titer globulin so as to sperm with non-isotopically tagged, block the non-specific globulin sites has not increased the sensitivity of the assay. Another problem with sperm is that variable numbers are lost during centrifugation and washing. In spite of their large size, sperm remain in the supernate in variable numbers. There is both species variation Separation by and individual variation in this discouraging phenomenon. filtration, even with non-radioactive-globulin saturated filters, results in a very large amount of radioactive globulin adhering to the filter,

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Rabbi& sperm 8 Ribonucleotide adjuvant (average of 3 rabbits, I Dr. Mengels lab) *Significantly higher

I

(0.01)

’

than

-

* pooled

4 3 + o 83

?;

adjuvant Menge’s

Seminal plasma 7 Ribonucleotide (I rabbit, Dr. lab)

2 2

0.97 (human sperm)

sperm)

adjuvant

2.4

2.3

2.9

1.8

+ 0.13

Human sperm 6 Ribonucleotide (I rabbit)

1.2

1.4 (human

adjuvant

adjuvant

Human sperm Freund’s adjuvant 5 (1 rabbit)

Saline solution 4 Ribonucleotide (2 rabbits)

Saline solution 3 Freund’s adjuvant (2 rabbits)

2 Ribonucleotide

‘~;j,i”I:;i;ii 1l-4+O.*’

Table II. Comparison of radioisotope assay tility of rabbits 3 weeks after immunization. and “d” (see text). 8 weeks experimental globulin plus pooled bucks (or human) spermatozoa Treatment Group (Test “b” ) /Rabbit sperm (Buck 44) 1

bucks

globulin

2.2

1.7 (human sperm)

2.3

I I

.5

2.2

I

2.1

X

1”

I

I

pooled

negative

doubtful

negative

negative

negative

plus

1

nega t i ve

negative

Macroagglutination (Kibrick)

I

sperm

l/l

l/l

l/I

l/2

2/2

314

2/4

zoa (Kibrick) B sperm, test

and fergroups “b”

infertile

(test

group

Successful

The isick.

I

I

I

pregnancy

“d”)

had a

doe was

The infertile doe severe infection.

Remarks IOne infertile doe was sick. The other infertile doe A_I. failed, she became pregnant on subsequent mating.

of spermat globulin/IO

Does with embryos vs. total does

bucks

/

I

I

with macroagglutination are mean values in mg

Pooled bucks globulin plus pooled bucks ‘(or human) spermatozoa (Test “d” )

results Units

Control (saline)

Seminal Plasma (vasectomized

28

Epididymal Sperm

"Results as reported from Dr. Menge's -:;+Results from our laboratory.

136

+

Semen

15

buck)

+

Semen

13

Fertility*

laboratory.

102,400

403,600

< 50

3,276,800

102,400

with

1,600

400

4

800

1600

Kibrick Agglutination t iterk

assay results interaction

Passive Hemagglutination t iter"

Comparison of radioisotope of sperm-antibody

Antigen Administered to rabbits"

III.

Globulin Sample Number

Table

parameters

1

1,024

<


1,024

512

Sperm Immobilization::

other

3.6

4.9

2.2

2.3

4.4

mg/lO

Sperm":

Radioisotope

assay8

CONTRACEPTION

despite extensive washing. promise, we are attempting lem by devising a soluble polyethylene test tubes.

Because

we believe

to avoid the sperm-antigen

radioisotope

whole-sperm that hopefully

assay

has

test-antigen probwill adhere to

Menge (1) reported that if seminal plasma from vasectomized bucks the doe rabbits responded with high was used as the immunizing antigen, moderate titers of sperm agglutinins, titers of hemagglutinating antibodies, Our results tentatively indicate no sperm immobilizing, and no infertility. that the radioisotope assay measures the same factor, or a closely associated The radioisotope as that measured by the sperm innnobilization test. factor, assay results correlated better with fertility than the hemagglutination assay did, but the radioisotope assay is not as sensitive as the hemagglutination assay for measuring sperm-globulin interaction. The most likely reason we failed to get significant titers of spermen. Menge injected binding globulins8was that we used a small dose of anti Our a total of 5 X 10 spermatozoa compared with our 6 X 10 3 spermatozoa. dosage was based on Braden’s (4) finding of a maximum of 4 X ‘06 sperm in Because Edwards (5) each of their two uterine horns following copulation. had difficulty immunizing rabbits against rabbit sperm without adjuvant, we decided to use adjuvant but kept the sperm numbers in the physiological range. We expected to find variable sperm-binding globulin in responses in different we concluded that natural sperm animals, but since none of the does responded, immunity would be rarely found in rabbits.

REFERENCES

1.

Menge, A.C . Use of polynucleotides isoantibod ies and infertility in 135:‘08-‘1 2 (‘970).

with

2.

Greenwood, F.C., Hunter, W.M., and ‘3”-‘abelled human growth hormone Biochem. J. 8g:“4-12’ (‘963).

3.

Kibrick, tion of 3:430-438

4.

Braden, female

5.

6.

64

S., Belding, D.L., antibodies against (‘952).

A.W.H. rabbit

Noyes, (‘969).

R.W.

Antibody

of

coitus.

Antigenicity Edwards, R.G. yolk after intravaginal J. Reprod. Fert. ‘:385-92

or

antigens

Proc.

Glover, of high

Sot.

to

Exp.

sperms

Aust.

J.

of rabbit intramuscular (‘960).

binding

of

in Biol.

the

genital SC’.

Med.

Fert.

tract

semen and of female

Steri’.

JANUARY 1973

of

the detecSteri I.

6:693-705

semen, bull injection

spermatozoa.

induce

Biol.

J.G. The preparation specific radioactivity.

and Merrill, B. Methods for mammalian spermatozoa. Fert.

Distribution after

seminal

rabbits.

in

the

(‘953).

egg rabbits.

20:43-49

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