Inhibition of in vitro fertilization of rabbit ova with naturally occurring antifertility agents

CONTRACEPTION

INHIBITION OF Iit vrz’m NATURALLY

FERTILIZATION OF RABBIT OVA WITH

OCCURRING

K. G. Gould, P. N. Srivastava,

ANTIFERTILITY

AGENTS

E. M. Cline and W. L. Williams

Department of Biochemistry, University of Georgia Athens, Georgia 30601, U.S.A.

ABSTRACT

Decapacitation factor (DF) and seminal plasma acrosin inhibitor have been shown to inhibit fertilization of rabbit ova in uivo. One peak obtained by DEAE chromatography of sperm acrosomes possessed a "true" neuraminidase activity, i.e. which released dialysable sialic acid from a substrate (Cowpers gland mucin). A second peak -rendered the sialic acid Neuraminidase reactive in the thiobarbituric acid assay but not dialvsable. has not been proven to participate in the fertilization process but will increase the resistance of the zona pellucida to dissolution by mercaptoethanol and bovine pancreatic trypsin. This work confirmed the antifertility effect of crude and pure DF in an in uitro system and showed that the peak from DEAE chromatography which possessed "true" neuraminidase activity inhibited fertilization while the other peak tested did not. It is suggested that sperm neuraminidase alters the tertiary and quaternary structure of the zona pellucida sialo-protein thereby rendering this layer less penetrable to spermatozoa. This may be one mechanism involved in the reduction of, or block to polyspermy.

Accepted

APRIL

for

publication

1971 VOL.

March

3 NO. 4

9,

1971

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CONTRACEPTION

INTRODUCTION

Several agents involved in the fertilization process have been extracted from seminal plasma and spermatozoa. Of these agents hyaluronidase (l), corona penetrating enzyme (CPE) (2) and a trypsin-like enzyme (3) apparently act to promote fertilization. Decapacitation factor (DF) and seminal plasma acrosin inhibitor (SPAI) have recently been shown to inhibit fertilization in vi00 (4). Two further fractions have been extracted from sperm acrosomes of the rabbit and bull both of which have neuraminidase activity. Due to differences in their mode of action one fraction has been designated sperm neuraminidase (SN) and the other neuraminidase-like factor (NLF) (5). Evidence for a physiological action of these extracts has not previously been presented. The demonstration of the effect of SN in prolonging the dissolution time of the zona pellucida of the rabbit ovum by mercaptoethanol and bovine pancreatic trypsin (6) promoted this investigation of the effect of SN, NLF and a more purified DF preparation than available heretofore on fertilization in vitro. Both DF and SN were shown to inhibit fertilization in vitro of both ovulated and follicular ova, confirming previous findings relating to in viuo fertilization in the former case and suggesting a physiological role of the latter in the fertilization process.

MATERIALS

AND METHODS

Ova were obtained by flushing the oviducts of mature New Zealand White does with 0.5 ml of the fertilization medium 13 hr after injection of 150 IU HCG IV. Two media were used in the in vitro system (Table I). Medium I is a modification of the defined medium of Brackett and Williams (7). Medium II contains 20% heated rabbit serum. Prior to use 5% CO, in nitrogen was bubbled throuoh the medium and overlaid with oaraffin oil. The medium was sterilized by-passage through a 0.45 P Millipbre filter and held under an atmosphere of 5% CO2 in nitrogen at 38'C. The final pH of the medium was between 7.6 and 7.8. The SN and NLF preparations being tested were prepared according to published methods (5,8), lyophilyzed prior to use and resuspended in sterile The crude DF of rabbit origin was prepared Medium I to a known concentration. by ultracentrifugation of seminal plasma. This preparation was further purified by digestion of the sediment with Pronase and chromatography of the dialyzable fraction on a Sephadex G-10 column, followed by chromatography on a powdered cellulose column (9). The DF samples were stored as a 10 mg/ml solution in distilled water and between 1 and 2.5 ~1 of this solution (lo-25 ug) added per ml of culture Medium II. Final volume of culture medium used in all experiments was 3.0 ml. Sperm concentration varied between 2.5 and 10 x lO'+/ml. Ova were incubated with SN and NLF for 30 min prior to insemination. Control ova were incubated

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TABLE I COMPOSITION

Medium

I:

OF DEFINED MEDIA

NaCl

8.0

KC1

0.3

CaC12

0.25

NaHzP04

0.105

MgClz

0.025

NaHCO,

2.7

Glucose

3.0

Bovine Serum Albumin

Na pyruvate at 5 x lo-'+ M. gassing

Medium

20%.

Penicillin

(BSA)

G at 50 u/ml.

3.0 After

- pH 7.8.

As Medium

II:

(mg/ml)

I but NaHCOs 1.5, no BSA and heated rabbit serum to

After gassing - pH 7.6.

I

1 1

in the culture medium alone for the same period. Ova were transferred to fresh Medium I prior to insemination thus reducing the concentration of NLF or SN by a factor of approximately 300. Spermatozoa were exposed to OF for 5-10 min prior to the addition of ova. Ova were transferred to 3 ml of Medium II 4.5 hr after insemination regardless of which medium was used during the first 4.5 hr. Ova were examined for evidence of fertilization 24 hr after The criteria used to establish fertilization were the presence insemination. of symetrical cleavage and staining of representative ova with acetocarmine. Ova were examined for continuina cleavaae UD to 36 hr post-insemination. In some experiments ova were follicle aspirated from pre-ovulatory follicles using a 1 cc syringe and a 0.5 inch 25 gauge needle with a steep bevel. The follicle wall was penetrated via the ovarian tissue (Fig. 1), a procedure found to eliminate haemorrhage during aspiration of the follicle The ova and associated cells were transcontents. ferred directly to the culture medium. Samples of SN and NLF were heated to 100°C for 5 min in culture medium without serum and tested for inhibitory effect on in vitro fertilization. __.._ In u,ivo fertilized ova flushed from the oviducts of capacitator dOeS were incubated with SN and NLF 50 ug/ml to ensure that neither agent inhibited cleavage per se.

rip

1.

RESULTS

Both preparations of DF used in these experiments were effective in inhibThe crude preparation was used at levels between iting in vitro fertilization. 90 and 400 ng/ml medium and was completely effective at all levels greater than

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90 pg/ml. The purified preparation was completely effective in inhibiting in vitro fertilization at levels over 10 pg/ml medium (Table II). TABLE II EFFECT OF CRUDE (CDF) AND PURIFIED (PDF) PREPARATIONS FACTOR ON FERTILIZATION IN VITRO

Experiment number

Amount added

*

Ova cleaved Total ova

Test

w/ml

OF DEcAPACITATION

I

CDF

200

o/71

II

CDF

400

O/5

III

CDF

150

IV

CDF

90

V

PDF

10

VI*

PDF

20

o/4

VII

PDF

20

O/6

VIII*

PDF

25

Control 3/41 -l/22

4.5%

317

12/20

O/7

3/5

l/3_

314 1 3/5 ---l/22

4.5%

60%

1

213

11/18

61%

3/6 3/4 :

Follicular ova.

Sperm neuraminidase (SN), under these conditions and at a concentration of 20 Pg/ml or greater reduced the rate of fertilization to almost zero (Table III). Heating the SN preparation to 100°C for 5 min destroyed the activity of the enzyme in the thiobarbituric acid assay (10) and allowed the fertilization rate to rise to a level statistically no different from that of the controls (Table III). Neuraminidase-like factor (NLF) at 100 ug/ml was not effective in preventing fertilization. NLF preparations heated as the SN preparations to 100°C for 5 min did not lose their activity in the thiobarbituric acid assay and were ineffective in altering the fertilization rate (Table IV). Neither SN or NLF inhibited cleavage of in Y~VO fertilized ova.

DISCUSSION The results obtained using the DF preparations confirm the antifertility effect of this substance as demonstrated in viva. The data emphasized the

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TABLE III EFFECT OF SPERM NEURAMINIDASE BEFORE AND AFTER HEATING ON FERTILIZATION IN VITRO 1

Concentration uglml

No. of expts.

Specific activity uM Sialic acid/min/mg

Ova cleaved Total ova

Test

Control

100

3

0.6

l/11

7110

36

1

0.3

O/4

2/3

25

4

0.6

o/17

12/20

20

1

0.4

o/3

3/6

50 (heated)

4

0.0

l/39

2.56%

24139

61.5%

8/15

53.5%

lo/16

62.5%

TABLE IV EFFECT OF 100 ug/ml NLF BEFORE AND AFTER HEATING ON FERJILIZAJION TN VITRO r Ova cleaved Total ova Test Unheated

8122

Heated

11/17

Control 36.4%

64%

11/19

58%

9115

60%

relationship between DF concentration and sperm count (4). Sperm count in this series (motile sperm/ml medium at time of insemination) varied between 2.5 and 10 x lo'+. In Experiment V (Table II), the sperm count was 10 x 104/ml, i.e. the ratio DF concentration/sperm was at its lowest level, 5 ug/5 x lo4 sperm. In experiments where DF completely inhibited in vitro fertilization, this ratio was higher, varying between 12.5 and 50 ug/5 x lo4 sperm. This concentration is lower than that routinely used in the in viva assay of DF activity (100 ug/105 sperm). This difference may reflect the relatively longer exposure of spermatozoa in the in ~itm, system to DF, due to the lack of uterine

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destruction

of DF (11) and hence continuous

exposure

of sperm in the system.

The discrepancy in results between SN and NLF can be explained by a consideration of the preparations used. The SN preparation, a single peak following DEAE chromatography, has "true" neuraminidase activity releasing dialysable sialic acid from the Cowpers gland mucin s bstrate. This SN preferentially cleaves the 2 + 6 ketosidlc linkage (5Y . The preparatlon of NLF used in this series of experiments renders sialic acid in the Cowpers gland mucin substrate reactive in the thiobarbituric acid assay but the product is not dialysable. A portion of the reactive sialic acid is dialysable, and this is attributed to contamination of the NLF with SN. Conversion of NLF to SN is a possibility not ruled out by experiments conducted to date, in fact, under special conditions this is known to occur (5). The apparent reduction medium is not statistically square.

in fertilization rate when NLF is included in the significant when analyzed by application of Chi-

The above data demonstrate an effect on fertilization of a sperm extract with neuraminidase activity. The mechanism of action is not known. Evidence has been presented that SN causes a chanqe in the tertiary and quarternary structure'of the protein moiety of the zona pellucida (6): This change reduces the oenetrabilitv of the laver bv spermatozoa (12) and increases its resistance to dissolution by bovine‘pancreatic trypsin: A similar change in resistance occurs at the time of fertilization in viuo. The SN is apparently very tightly bound to the sperm, probably on the inner acrosomal membrane. This fact, together with the presence of sialic acid residues in the zona pellucida (13) suggests an analogy with the demonstrated interaction of Viral neuraminidase and the membrane of a potential host cell during the attachment of a virus particle prior to invasion (14). This line of thought suggests the interesting possibility that SN may function initially to promote and later to inhibit one part of the fertilization process. An initial interaction between SN and the zona" sialic acid promotes an adherence of spermatozoa to the ovum, a necessary prerequisite to penetration by the spermatozoon of this layer. At the same time the SN acts on the sialo protein of the zona and renders it, at least in the rabbit, gradually less susceptible to dissolution by a proteolytic sperm enzyme, thus retarding the penetration of the zona by sperm, or actually reducing the number of spermatozoa penetrating the zona.

ACKNOWLEDGMENTS

The research upon which this publication is based was performed pursuant to Contract No. NIH 69-2103 and Career Development Award 2K3 GM4831-09 with the National Institutes of Health, Department of H.E.W. Partial support was also received from the Ford Foundation and Contract No. NIH 70-2147

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REFERENCES

1.

Austin, C. R. Capacitation and the release of hyaluronidase J. Reprod. Fert. 3, 310-317, (1960). spermatozoa.

from

2.

Zaneveld, L. J. D., Srivastava, P. N., and Williams, W. L. enzyme that disperses the corona radiata and its inhibition tation factor. Biol. of Reprod. 2, 363-368, (1970).

3.

Zaneveld, L. J. D., Srivastava, P. N., and Williams, W. L. Relationship of a trypsin-like enzyme in rabbit spermatozoa to capacitation. J. Reprod. Fert. 20, 337-339, (1969).

4.

Zaneveld, L. J. D., Robertson, R. T., Kessler, M., and Williams, W. L. Inhibition of fertilization in viva bv oancreatic and seminal plasma trypsin inhibitors. J. Reprod. Fert.- In press.

5.

Srivastava, P. N., Zaneveld, L. J. D., and Williams, W. L. Mammalian Biochem. Biophys. Res. Comm. 39(4), sperm acrosomal neuraminidases. 575-581, (1970).

6.

Gould, K. G., Zaneveld, L. J. D., Srivastava, P. N., and Williams, W. L. Biochemical changes in the zona pellucida of rabbit ova induced by fertilization and sperm enzymes. Proc. Sot. Exp. Biol. Med. 136, 6-10, (1971).

7.

Brackett, B. G., and Williams, W. L. Fertilization of rabbit ova in a defined medium. Fert. and Steril. 19, 144-155, (1968).

8.

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A sperm by decapaci-

E. F., and Srivastava, P. N. Chemical composition of the J. Reprod. Fert. 9, 47-60, (1965). of ram spermatozoa.

10.

Warren, L. The thiobarbituric Chem. 234, 1971, (1959).

11.

Abney, T. O., and Williams, W. L. Inhibition of sperm capacitation by Biol. intrauterine deposition of seminal plasma decapacitation factor. of Reprod. Z(l), 14-17, (1970).

12.

Soupart, P., and Clewe, T. H. Sperm penetration of rabbit zona pellucida inhibited by treatment of ova with neuraminidase. Fert. and Steril. 16, 677-682, (1965).

13.

Soupart, P., and Noyes, R. W. Sialic acid as a component of the zona pellucida of the mammalian ovum. J. Reprod. Fert. 8, 251-253, (1964).

14.

Gottschalk, A. The influenza 377-378, (1958).

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