RAPID COMMUNICATION

ZIPGRAM

EVIDENCE FOR THE ROLE OF A TRYPSIN-LIKE ENZYME IN THE HAMSTER SPERM ACROSOME REACTION (1)

STANLEY MEIZEL AND CHUNG W. LUI Department of Human Anatomy, School of Medicine, University of C a l i f o r n i a , Davis, California 95616 ABSTRACT Acrosome reactions occurring in v i t r o i n hamster sperm capaci t a t e d by bovine fol1 i cul a r f l uid were seyerel y inhibited by four s y n t h e t i c trypsin i n h i b i t o r s and by Zn t. Three polypeptide trypsin i n h i b i t o r s and a s y n t h e t i c c h j m t r y p s i n i n h i b i t o r did not inhibig the acrosome r e a c t i o n , and Ca t overcame the inh i b i t i o n by Zn +. These r e s u l t s suggest t h a t a trypsin-like enzyme (possibly a c r o s i n ) plays a r o l e in the acrosome reaction. The acrosome reaction, an event e s s e n t i a l t o mammalian sperm penetration of the zona pellucida, i s a progressive fusion, v e s i c u l a t i o n , a n d l o s s of t h e outer acrosomal membrane and the adjacent sperm head plasma membrane.

These

changes occur in sperm t h a t a r e near o r in the egg cumulus oophorous and t h a t have undergone those physiological changes known as capaci t a t i o n (Bedford, ' 7 0 ) . Capacitation and the acrosome reaction can a l s o be induced i n v i t r o by incubation of hamster sperm with bovine f o l l i c u l a r f l u i d (Yanagimachi, ' 6 9 ) . Experimental evidence suggests t h a t the sperm trypsin-1 ike enzyme acrosin ( E C 3.4.21.10) i s involved in zona penetration (Stambaugh e t a l . , ' 6 9 ) and t h a t

acrosin i s localized on the sperm equatorial segment and/or inner acrosomal membrane (Brown and Hartree, ' 7 4 ) .

Hamster and r a b b i t cauda epididymal sperm

acrosins a r e present mainly a s the zymogen precursor proacrosin (Meizel and Mukerji , '75a,b).

Hamster acrosin prepared by autoactivation o f proacrosin

can hydrolyze the zona pellucida of hamster eggs (Meizel and Mukerji, manuscript i n preparation ) . 'I 37

Schuel e t a l . ( ' 7 3 ) have proposed t h a t t r y p s i n - l i k e enzymes a s s i s t i n the discharge of secretory granules and then p a r t i c i p a t e i n other specialized c e l l functions.

I t has been postulated t h a t acrosin could be involved in vesicula-

tion d u r i n g the acrosome reaction (Gordon, ' 7 3 ) . The present paper describes the f i r s t experimental evidence which suggests t h a t a trypsin-like enzyme (possibly acrosin) plays a r o l e in the acrosome reaction of a mammal i an spermatozoon. MATERIALS AND METHODS

Benzamidine

Chemicals; p-aminobenzamidine

. 2HC1

-

HC1 was obtained from Eastman Organic

and a-N-tosyl -L-lysine chloromethyl ketone

'HC1 (TLCK) were purchased from Bachem Co.; soybean, lima bean, and egg white trypsin i n h i b i t o r s , a-N-tosyl-L-phenylalanine chloromethylketone (TPCK), pnitrophenol and p-ni trophenyl-p-guanidinobenzoate from NBC.

*

HC1 (NPGB) were obtained

Eppendorf micro t e s t tubes (1.5ml) were purchased from BioRad.

Bovine f o l l i c u l a r f l u i d was obtained, stored, and detoxified by heating t o 56'

C p r i o r t o use as described by Yanagimachi ( ' 6 9 ) except t h a t the f l u i d

was heated f o r 1 hr.

Cauda epididymal sperm were obtained from mature golden

hamsters as described by Yanagimachi ( ' 6 9 ) . and Meizel were then used:

The following methods of Cornett

(1) sperm were allowed to disperse f o r 10 min a t

room temperature in a modified Tyrode's solution containing 25 mM sodium bicarbonate and 140 units/ml of sodium p e n i c i l l i n , adjusted t o pH 7.4 with 100% CO,;

( 2 ) sperm dispersion took place in s t e r i l e disposable p l a s t i c dishes in the ab6

sence of mineral o i l ; ( 3 ) sperm concentrations ranged from 6 t o 7.5 x 10 /ml; ( 4 ) incubations o f hamster sperm in bovine f o l l i c u l a r f l u i d were carried out

in the absence of mineral o i l in 1.5 ml polypropylene t e s t tubes (rinsed and autoclaved p r i o r t o use); ( 5 ) three volumes of f o l l i c u l a r f l u i d were diluted w i t h two volumes of the modified Tyrode's solution; ( 6 ) the pH of the diluted

f l u i d was adjusted t o 7.4 with 100% CO,; 138

( 7 ) 50 pl of the f l u i d followed by an

equal volume o f sperm suspension were added t o t h e i n c u b a t i o n tubes; ( 8 ) two h o l e s were made i n i n c u b a t i o n tube l i d s w i t h a 25 gauge needle, and t h e l i d s closed; ( 9 ) t h e tubes were i n c u b a t e d a t 37'

C i n a humidified incubator w i t h

5% C02 i n a i r . I n a l l cases t h e pH o f i n c u b a t i o n m i x t u r e s were m a i n t a i n e d a t 7.5-7.6 throughout t h e i n c u b a t i o n . mosmol

The o s m o l a l i t y o f a l l i n c u b a t i o n m i x t u r e s was 315

.

Most i n h i b i t o r a d d i t i o n s were made by adding 5 1~1o f t h e p a r t i c u l a r compound d i s s o l v e d i n m o d i f i e d T y r o d e ' s s o l u t i o n t o t h e 100 1~1o f i n c u b a t i o n mixture.

F i v e 111 of ZnC1,

p l u s CaC1,

o r ZnC1,

alone d i s s o l v e d i n 1 mM HC1 o r 5

p1 o f NPGB o r p - n i t r o p h e n o l d i s s o l v e d i n 3% d i m e t h y l s u l f o x i d e (DMSO) i n H20 were added t o t h e i n c u b a t i o n m i x t u r e .

F i v e 111 o f TPCK d i s s o l v e d i n a b s o l u t e

e t h a n o l was added t o 1 m l o f d i l u t e d f o l l i c u l a r f l u i d , and 50

111

c o n t a i n i n g f o l l i c u l a r f l u i d s o l u t i o n was i n c u b a t e d w i t h 50

of the o r i g i n a l

sperm suspension.

pl

o f t h i s TPCK

The f i n a l c o n c e n t r a t i o n s o f e t h a n o l and DMSO were 0.25% and

0.15% r e s p e c t i v e l y .

A method devised by C o r n e t t and M e i z e l was used i n o r d e r t o e s t i m a t e t h e percentage o f sperm m o t i l i t y and a c t i v a t i o n :

(1) a tube's e n t i r e contents

were t r a n s f e r r e d by a pre-warmed p a s t e u r p i p e t t e t o a p o s i t i o n underneath a cover s l i p supported by dots o f v a s e l i n e - p a r a f f i n on a pre-warmed g l a s s s l i d e and s t u d i e d by phase c o n t r a s t microscopy a t 125x m a g n i f i c a t i o n ; ( 2 ) d u r i n g these m i c r o s c o p i c o b s e r v a t i o n s , t h e s l i d e was m a i n t a i n e d a t 37' C by a Sage a i r - c u r t a i n microscopic incubator.

A c t i v a t e d sperm were c h a r a c t e r i z e d by an extreme v i g o r -

ous w h i p l a s h - l i k e f l a g e l l a movement (Yanagimachi,

'74).

The percentage o f 100 h i g h l y m o t i l e sperm which had undergone t h e acrosome r e a c t i o n was then determined w i t h these same s l i d e s a t room temperature u t i l i z i n g phase c o n t r a s t microscopy a t 400-5OOx m a g n i f i c a t i o n (Yanagimachi, ' 6 9 ) .

139

RESULTS When control hamster epididymal sperm were incubated with bovine

f o l l i c u l a r f l u i d a t 37' C, no activation was observed u n t i l 2.5 hrs.

The f i r s t

acrosome reactions occurred a f t e r 3 hrs, and the reaction reached i t s maximum a t about 4 . 5 hrs.

A t t h a t time, 60-80% of the sperm were motile and 40-60%

were activated. When two competitive trypsin i n h i b i t o r s benzamidine and p-aminobenzamidine (Mares-Guia and Shaw, '65) were added t o the incubation media, there was a drast i c inhibition of the acrosome reaction ( f i g . 1 ) . These i n h i b i t o r s did not a f f e c t the sperm's m o t i l i t y o r a c t i v a t i o n during the incubation.

The active

s i t e trypsin t i t r a n t s , NPGB (Chase and Shaw, ' 7 0 ) and TLCK ( a l s o an i n h i b i t o r of papain.

Whitaker and Perez-Villasenor, ' 6 8 ) , severely inhibited the acro-

some reaction ( f i g . 1 ) . Neither of these i n h i b i t o r s had any e f f e c t on m o t i l i t y , b u t they did i n h i b i t sperm activation by 20%. When added a t the same concentra-

t i o n s as TLCK, the chymotrypsin and papain i n h i b i t o r TPCK (Whitaker and PerezVillasenor, '68) did n o t i n h i b i t the acrosome reactions.

ZnC1, (2.5 x lO-'+M)

a l s o greatly inhibited the acrosome reactions ( f i g . l ) , b u t when CaC1, (2.5 x lO-3M)

was added together with ZnCl 2 , the acrosome reaction was not inhibited.

Polypeptide trypsin i n h i b i t o r s from soybean, lima bean, and egg white a t f i n a l concentrations of 1 mg/rnl did not i n h i b i t the acrosome reaction.

FIGURE LEGEND 1 The percentage of acrosome reactions a t d i f f e r e n t incubation times in the absence o r presence (during the S n t i r e incubation) of seve r a l synthetic trypsin i n h i b i t o r s or Zn +. The followin s mbols control; & - - A , benzamidine (7.5 x 10 M are used: X-X, O - - - o , p-aminobenzarnidine ( 5 x 10- 4M)L; m-B, TLCK x 10-5M); ~4 NPGB,( 5 x 10-5M); 0----0, ZnC12 ( .5 x lO-'+M). The control value represents the range and mean of a l l experiments. For the experimental values, every v e r t i c a l l i n e represents the range and mean of 3 determinations using d i f f e r e n t f o l l i c u l a r f l u i d s .

-Y Y

1s

140

90 T

80 70 60 -

50 40 -

30 -

T

/

20 -

101

3:OO

1

3:IO

I

3:20

I

1

I

I

I

I

3:30 3:40 330 4:OO 440 INCUBATION TIME (HRS:MIN)

4:20

4:30

Although n o t shown in figure 1, benzamidine (7.5 x 10m4M)o r NPGB (7.5 x 10-5M) a l s o inhibited the acrosome reaction by 50-70% when added as l a t e as 2.5

hrs a f t e r beginning of the incubation. f e c t sperm a c t i v a t i o n .

In these experiments, NPGB did n o t a f -

P-nitrophenol (7.5 x 10m5M), a product of NPGB hydro-

l y s i s did not i n h i b i t the acrosome reaction when added a t 2.5 hrs. DISCUSSION An obvious question which must be asked i n analyzing these r e s u l t s i s whether or not the inhibition of t r y p s i n - l i k e a c t i v i t y by the e f f e c t i v e i n h i b i t o r s i n t e r f e r e d with capacitation o r the acrosome reaction? Obviously interference with capacitation would a l s o i n h i b i t the acrosome reaction. Since sperm activation only occurs in capacitated hamster sperm (Yanagimachi, ' 7 4 ) , the absence o r low l e v e l s of inhibition of a c t i v a t i o n by the synthetic

i n h i b i t o r s s u g g e s t s t h a t capacitation was not i n h i b i t e d . The synthetic i n h i b i t o r s inhibited and the polypeptide i n h i b i t o r s f a i l e d t o i n h i b i t the acrosome reaction a t i n h i b i t o r concentrations s i m i l a r t o those which i n h i b i t hamster acrosin (Meizel and Mukerji, '75a and manuscript in preparation).

One i n t e r p r e t a t i o n of these r e s u l t s i s t h a t the low molecular weight

(156-336) synthetic i n h i b i t o r s b u t not the high molecular weight (8300-28,000) polypeptide i n h i b i t o r s entered the acrosome.

I f i n t r a c e l l u l a r inhibition oc-

curred, acrosin was probably the enzyme inhibited.

Another trypsin-1 ike enzyme

has not y e t been detected in hamster sperm. A1 though we have not concl usively ruled out the possi bil i t y of a non-sperm e x t r a c e l l u l a r trypsin-li ke enzyme being involved, preliminary assays have n o t detected such an enzyme in hamster epididymal f l u i d o r detoxified bovine f o l -

1icular fluid.

In addition, Yanagimchi (personal communication) found t h a t

pancreatic trypsin does not accelerate guinea pig acrosome reaction induction, and Gwatkin and Hutchison ( ' 7 1 ) reported t h a t trypsin did n o t capacitate hamster sperm.

142

In the present paper, we have a l s o shown t h a t Zn

'+

inhibited the acrosome

Ca

*'

i s known t o stimulate

reaction and t h a t Ca

2t

overcame t h i s i n h i b i t i o n .

the acrosome reaction of guinea pig and hamster sperm (Yanagimachi, '74 and Talbot ' 7 5 ) .

Meizel and Mukerji ( ' 7 5 a ) have reported t h a t hamster proacrosin

autoactivation and acrosin a c t i v i t y were stimulated by Ca

Zn '+.

2t

and i n h i b i t e d by

Thus, the e f f e c t s of these ions on the hamster sperm acrosome reaction

might be due a t l e a s t in part t o t h e i r e f f e c t on acrosin and proacrosin. Future studies will be undertaken t o c l e a r l y e s t a b l i s h (1) the i d e n t i t y o f the trypsin-like enzyme involved i n the hamster sperm acrosome reaction and

( 2 ) whether i t s r o l e i s d i r e c t o r i n d i r e c t (e.g. a c t i v a t i o n of another enzyme).

ACKNOWLEDGMENTS The authors w sh t o thank Dr. R. Yanagimachi and Dr. B . Bavister f o r t h e i r demonstration of capacitation techniques and stimulating d i sc uss i on s concern i ng the acro some reaction.

We a l s o thank o u r colleague Dr

S.K. Mukerji f o r h i s help with prel minary enzyme assays.

LITERATURE CITED Bedford, J.M. 1970 Sperm capacitation and f e r t i l i z a t i o n i n mammals. Biol. Reprod., Supplement 2 : 128-158. Brown, C . R . , and E.F. Hartree 1974 Distribution of a t r y p s i n - l i k e proteinase i n the ram spermatozoon. J . Reprod. F e r t . , 36: 195-198. Chase, T . , J r . , and E. Shaw 1970 In: Methods in Enzymology. Vol. 19. G . E . Perlman and L . Lorand, eds. Academic Press, New York, p.20-27. Gordon, M . 1973 Localization of phosphatase a c t i v i t y on t h e membranes o f the mammalian sperm head. J . Exp. Zool. , 185: 111-119. Gwatkin, R . B . L . , and C . F . Hutchison 1971 Capacitation of hamster spermatozoa by a-glucuronidase. Nature, 229: 343-344. Mares-Guia, M., and E. Shaw 1965 Studies on the a c t i v e center of trypsin. J . Biol. Chem., 240: 1579-1585. Meizel , S. , and S.K, Mukerji 1975a Hamster sperm proacrosin and acrosin. Abstracts of t h e Annual Meeting of the Society f o r the Study of Reproduction, p . 26. Meizel, S . , and S.K. Mukerji 1975b Proacrosin from r a b b i t epididymal spermatozoa: P a r t i a l p u r i f i c a t i o n and i n i t i a l biochemical charact e r i z a t i o n . Biol. of Reprod., 13: 83-93. Schuel, H . , W.L. Wilson, K. Chen and L. Lorand 1973 A t r y p s i n - l i k e proteinase localized i n c o r t i c a l granules i s o l a t e d from u n f e r t i l i z e d sea urchin eggs by zonal centrifugation. Role of the enzyme i n f e r t i l i z a t i o n . Dev. Biol. , 34: 175-186.

143

Stambaugh, R., B.G. Brackett and L . Mastroianni 1969 I nhibition of -i n v i t r o f e r t i l i z a t i o n o f r a b b i t ova by tr ypsin i n h i b i t o r s . Biol. Reprod. , 1: 223-227. Talbot, P . 1975 The e f f e c t o f ions on the acrosome r eaction. Abstracts, Annual Meeting of the Society f o r the Study of Reproduction, pp. 28-29. Whitaker, J.R., and J. Perez-Villasenor 1968 Chemical modification of papain 1. Reaction with the chloromethyl ketones of phenylal a n i n e a n d lysi ne and w i t h phenylmethylqulfonyl f l u o r i d e . Arch. Biochem. Biophys., 124: 70-78. Yanagimachi , R. 1969 In vi t r o acrosome reaction and capaci t a t i o n of go1 den hamster spermatozoa by bovine f o l l i c u l a r f l ui d and i t s f r a c t i o n s . J . Exp. Zool., 170: 269-280. Yanagimachi, R., and N. Usui 1974 Calcium dependence o f the acrosome reaction and a c t i v a t i o n of guinea pig spermatozoa. Exp. Cell Res., 89: 161-174. REFERENCES

1 This research has been supported by N I H Grant HD-06698 and in part by NIH Grant HD-07893.

144