Purification and characterization of an anti-sticking factor from goat epididymal plasma that inhibits sperm-glass and sperm-sperm adhesions

114

g~/t6a/m a ~

Ate. 9~)!O ~

11t--122 Elsm~r

BILA23O94

Purification and characterization of an anti-sticking factor from goat epididymal plasma that inhibits sperm-glass and sperm-sperm adhesions Nabina Roy and Gopal C. Majumder

~ . ~ , e d 3~ October 1 9 ~

Key~:

~tistifi~g faet~, spe~ a d ~ i ~ ( ~

q~al

t~)

~ ~ fm~ (~-0 d m s~ewed ~ am~i~ ~ ~ ~ee ~ s p e n m t e ~ m te ~ m ~ ~ from g ~ t e l ~ i , tymal l / m m a aml d, m a e t e r l z ~ Tl~e f a e t ~ was t,mified ~ m ~ x . . ~ d e J e l d aml sb0wed a s i q k i,m t ~ t vahte el' ASF-I mere a p l a ~ 47 Ikl~ am]l ~2S S. ASF-I a / a ~ d i a M slmw~l m ~ d y m m e m ~ i t b s l i d ~ g ae/~ey when a l ~ n ~ . (~OYod ~ e I~K/Sl~.~m~oma ~ ' e 1 , 8 ~ , ~ d f ~ m adhes~m m s l a ~ a ~ I ~ s ~ w e d a ~ ~ e~ ~ spec~,~ty. Se~M~ w~h w ~ , s ~ and ~ ' ~ m s d , ~ u m s m t ~ / h a t buth ff~e s,~ar a ~ l ~ imps ~ ~ m o ~ c ~ e are ememial f~" its a n ~ . s e ~ k ~ k i b e s . F.~dm,~e has h e m I R s e m e d to suRm~ ~ ~ ~ ~ ~ s ~ f a e e e l Slmrm ~ spetffie AS~-I receptms Ihat l ~ d to n S l ~ ASF aml m-~am, edl adhesim ~ ~ ASF-I also s b m ~ l la]~ a f ~ i t ~ |e~ ~ 6 m l g tgglugimaim~ d cmlam-epklidymal ~ T I ~ AS~ agGvlly w ~ fmmd ~ be d i a n ' ~ a d ~ ~1 the tismos m ~ d --.a ~ specWg ~ t ~ ~ m~lb l ~ l m r ~. ~ o d ~ ~ ~ ~ fissue~ ~ne resutts ~ that ASF may play an i ~ r m a t I ~ o ~ d role I ~ s e r ~ g as a sl~eci~ ~ l ~ e r ~ cell-sul~tratum a~l eeB--cell atlteslem.

It is ge~endly recognized that spermatozoa, like most other types of cel~ adhere to glass sudaces. Haemocytometer couming chambers age in wide use for subjective and objective assessm~ts of sperm modlity [1]. Howev~, the conm~out~q0n of the process of sperm sticking to the glass s u r f ~ e of the haemocytometer counting chamber has largely been oveJooked by ~he earlier investigators. Stephem et al. [2] have obse~ed that bovine immature caput-spen~utmzoa stick to the glass sudac~ of the counting dmmber during motility assays and this phenomenon of cell adhesi~m may pose as a great artifact for the estimatio~ o f sperm motility. It has recently been reported from our laboratory that

Atx4~reviatic~s:EP. epidid3nmal ip~sma; /~F, amJ.s~,'~n~_ f~m~;, PMSF, phe~yhue~yt~pheayJ ~ d e ; RFS,me.lied Rinses setudon. ~ : G.C. M a l a d y . g n d ~ In.irene of Chemical Biolo~. 4, ~ZajaS.C. MulE.d: Road, Jadavpug. Cal~sta 700 032. India-

goat matm'e cauda-spermatozoa also adhere to t l g h a e m o ~ 0 ~ e t e r glass smface [3L ~ of the glass could not pr~-e~t dte ce.~ from s t l c t i K to it. Cauda Slmmatezo~ once they had adhered to glass. could not be dissociated by flte~hing with high c o w centrations of salt (1 M NaCrl) or q~Mid3m~l plasma, intimating that ceil-glass adhesion is tight and ~wevetsible [3]. It is thus n ~ c a ~ y to identify a potent factor that wal prevent with high specificity and affiaity sperm a d h e ~ o n to 81ass, so that i t c a n be used to imlao.~e the

e x i s t i ~ motility assays by eliminating the ~ t y cen-stick~ artifa~

of

O a r pr(4iminary studies [3] have

provided evidence for the occurreace o f heat-stable anti-sticking factor(s) (ASF) in goa: EP that strongly inlu'bit adhesion of mature c a u ~ i d i d y m a l spermatozoa to glass. ASF is a protein, since its activity is destroyed by the action of trypsin. Bivaleat metal ions, a s M g 2+ and C a z+, had no effect ~

the A S F

activity of EP. This study r q ~ l s tl~ ~ t k m of an ASF from goat EP to apparent homogeneity. Stone of the physical and biochemlcal chagactedst~ o f the factor have been investigated.

0304-41651q19/Y~03~0© | ~ 9 Else~er Science Pabl~'~'s B.V. (Biomedical ~ )

115 ~

alal Methe~

Pel3~e~lene Blycol),DEAE-ccBulo~ methyl

a-

PMSF, z-fucosidase, , 8 - N - ~ i , - i . k l a ~ , a-mnn~micla~ neuraminida~ i d a ~ return, myoglob~ b u ~ e m r m a l b u m ~ Bhao~bu~ o v a t , - - m ; trypsin (twice c ~ d ) , c b y m o u 3 ~ matin, ~ phe~ aad F¢o¢400 were obtained from Sisma, SC Lonls, MO, U.S.A. Cert. canavalin A-alpJro~ was a product of H ~ Cahmua. Naaz~! was sup,jp~ed by the Bhabha Atomic 1P,~arch Canue, Bombuy. Iodobead was obtained from Pierce, P,~tfo,'d, IL, U.S.A. Spermatoana were obtained frem fr--,=,hgoat cauda Highly motile s p e r ~ - , ~ _ w ~ e extracted at room perature O l :I: I ° C ) from the op~IMymidm in a mod-

ificdBin~"~'Ssohmfion(RPS medhnn" 119 m M NaCI/5 mM KCV] mM CaO~q2 mM MsSO,,~0 mM ~cose/16.3 mM pmas~nm ~ t e (pH 6.9)/50 units/ml peaicillin). Numbe~ of q~mnato~m ia the samples were dea:rmlaed with a ~ . The sperm preparations wece highly pine, as judged by light mkmscopy, and c'~utained le~ than 2~ brokm c~

dm~d ce~ For the preparation of EP, the freshly extracted sperm preparation was c a n u i f u ~ l at 800 × ~ fc~ 10 rain- Tbu resulting ~,wemate was c~td~Lt~d a ~ i n at 18000 x g to obtain cen-fme EP. EP was stmed at - I O ° C with PMS~F O mM). A n t i - ~ f i n g activity of EP and its fractions was measured by the procedure reported eadier 13l with mino~ ~ An alMuot of tha sporm ~ lion (25 pl cc~taln;ng approx. 0.5-106 gg~J~) i~:ubated at teom Icmpcrateg ( 3 1 + 1 ° C ) with or without a specified amount of anti-stickin~ activity in a lotal voL of 0.5 ml of RPS modh,m A portioa of the cdl mspoas/on was thin injected into the ha~acoya~metew and th~ inltla] c~] number was cot~t~d (CT) t m d ~ a phase-contrast microscope at x250 magnification. A f t ~ 5 rain incubation, the conating chamber was llushed with an ~ of RPS medimn (3 x 100 ~1) to nnnove tha celb that were in saspemion, i . ~ those cells ~hat did mot sllck the glass. The tint.him, was done by injection of the buffer with a 100/d aatopipette into the counting chamber from the front side of the hacmoeytomemr, taking case not to disturb the cover-dip. The cells that were left after flushing in the dmmber were rhea couated and ~ valuas (Cs) represent the cells that adhenxl firmly to the 81ass surface of the haemocytomet~r. The pet¢cotage of cens that did not adlu~e to the glass surface of the hacmocytometer chamber was

ca~datod as [(CT - C-~)/CT] x 100. A unit of activity of ASF was defined as the amount of the factm" that prevents sticking of 10~ of the celk under the standard assay conditions. Systems lacking exogcnons anti-stickinB activity seTved as the blank in all assays and the blanks showed a very low amount of anti-sticking activity (O-0.3 U).

P t ~ f ~ n o/ ASF.Z C4mconav~n A-affinity chronmtography. EP (approx. 3.05 m~ prmein/ml) was concentrated to n great extem (api~ox- 25.4 mg protein/nd) with poly(ethylene glycol) and then dialysed against 20 mM T r b - H O (pH 7.2)/1 taM CaClJl m M MnCIJ0.6 M NaCI ('ot~fer l) before be/as subjected to affinity chromatography on a CAm A-agasose column (1.8 x 15 cm) previously ¢quilibrated with buffer I. The column was washed with 150 ml of the equilibrating baffer. The anti-sticking activity was ehtted first with 150 ml of buffer 1 containing 30 m g / m l of methyl a-D-mannosid¢ and t h ~ with 90 mt of 50 m M glycine-HO (pH 3.0)/1 M NaCI/15 m~/ml methyl a-D-mannoside. This Con-A-eluate was neutral. ized, concentrated by Amionn ultraflltration technique using PM-30 membrane and dialyr~l against 10 mM Tds-HCI (pH 9.0). DEAE.~Iulme chromatography. The dialy'~d ASF preparation was ~plied to a D ~ u l o s e colunm (1.0×15 a n ) equifibrated with 10 mM Tris-HCI (pH 9.0). After passable of the sample, the column was washed with 30 ml of the cquigibratingbuffer. The peak of the activity (ASF-I) was eluted with 30 ml of 0.1 M Tris-HCI (pH 9.0). The column was thea eluted successively with 25 ml e ~ h of Trls-HCI (pH 9.0) havin~ concentrations of 0.2, 0.3, 0.5 and 0.7 M and the eluates were discarded. ASF-II was finally eluted from the column with 25 ml of 1.0 M Tris-HCl (pH 9.0)/1.0 M NaCI. The fractions CODl~nla~Ab~-I and -II activities were concentrted by ultrafiltrafion through Amicon PM-30 membrane and then ¢quih'brated with 5 mM potassium phosphate (pH ?~0). Acrylamide-Sephadex gel electrophoy~$i$. ASF-I was purified further by using a novel electrophoresls system consisting of polyacrylamlde and Sephadex G-200 gels as the matri¢ The resolving (7~ acrylandde) and stacking (3~ acrylamlde) polyacrylan~de gels were pr~ased according to the method of Laemmll [4], except that SDS was omitted. A layer of preswogen Sephadex G-200 (4 x 0.6 cm) was incorporated in the gel tube with resolvin~ gel at the top (4 x 0.6 cm) and the bottom (2 X 0.6 era) (as shown in Fig. 1A). The bottom polyacl'ylamlde ~ BaV¢ SllppOl~ gO the S~phadex layer. Sephadex was dispersed in 0.3?5 M Tris-HCI (pH 8.0)/10~ sucrose. ASF-I was lyophilized ~nd dissolved in 250-300 pl 0.06 M Tris-HCl (pH 6.?)/10~ 81ycerol/l~ fl-

116 mercaptoethanol (approx- 8 mg protein/hal). The gels were pruelectrophoresed for 1 h at 5 ° C whh a current of 5 m A / g e l befo~ loading each gel tube with approx. 400-500 p g protein of ASF-I pr~paratior. Electrophoresis towards the anode was canied out for 2.5-3 h with a current of .~ m A / g e l using Bromoph~nol blue as the tracking dye. The bottom polyaorylamlde gel was then removed by pushing the gel from the top with a plunger. The Sephadex layer comalning the ASF-1 band was then pushed out and collected in a small beaker. The Sephadex G-200 materialswere pooled from four or five gel tubes and u'ansferred to a small Phermacia column (inner diameter 9 nun). The anti-sticking activity was duted with 5 ml of 5 mM pomssinm phosphate (pH 7.0)/1 mM PMSF. The fraction was concemrated with 40% glycerol/1 m m P M S F / 5 mM potassium phosphate (pH 7.0) and preserved in the same buffer at 10°C. n2Sl-labellingof ASF-I. ASF-! was radioiodinated by the method of Markwell [5] with some minor changes. Ovalbumin, which has no non-specific anti-sticking activity, was used as !1~ carrier protein instead o f BSA. ASF-I (2 ~g) was incubated at 31°C for 10 rain with 5 mmol of sodium phosphate (pH 7.2), 0..5 mCi of Nat251 and one iodobtad in a tmal voL of approx. 100 pL The Z~l-lahelIed ASF w ~ then separated from the free iodide by molecular sieving on a column of Sephadex G-25 (0.8 x 21 cm) using PBS as the duting buffer [5].

Assay of binding of IzSI-ASF-I by spermatozoa. The standard assay medium conlained 5 ng of 125I-A~F-I (containing 5-103 cpm) and intact spermatozoa ( i - 10 ~) in a total vol. of 0-5 ml of RIPS medium. Incubation was carried out at 3 7 ° C for 5 rain and the binding reaction was arrested by a d ~ a g poly(ethyleneimine) (100 t~g/ml), a polycatiun that caused instant aggluti~zfion of cells [6]. The cells were immediately sedimented by centtifugafion at 450 x g for 5 rain, the cell pellets were washed twice in RPS medium and then counted for radioactivity in a gamma counter. For the estimation of the blank values, EP (1-~ m g protein) was added to each assay to cause complete displacement of 1251-ASF-I, specifically bound to the cells, by the unlab¢lled ASF-I p r e s t o in EP. The blank tubes gave values pdmasily due to non-specific binding of the labelled ASF to the cells. The binding data were corrected for the blank values.

The fractions were counted for radioactivity in a gamma counter to localize the peak of ASF-L The peak of the marker protein was d c t ~ by estimating the prorein contents of the fractions.

Protein ¢~timatio~ U n l ~ othegwise specL~d, tl~ proWin contents of the samples were estimated according to the methed of Lowry et at. [7] m~n8 bovine serum albumin as standard. Protein oanvemratim~ of the samples containing 81ycerol were e~timated by the method of Bensudoun and Weinsrein [8]. which involves pxec~Dimtinn o f prowin with trichlorozcetic acid and sodium deoxycholale prior to the coIorimetric assay of prmeln. P.es~s

Purification of A S F The s u m m a ~ of the purificatiun of the ansi-sdcking factor h ~ been fJu3wn in Table L ASF binds with high affinity to the Cou A-agarc~e colmnn and the activity is eluted slowly first with methyl a-mannoside {30 rag/nil) and then with the mannoskle containing the acidic glycin~HCI buffer (pH 3.0). By the Con A-affinity chromatographic step the recovery of ASF activity was nearly 150%, suggesting that there had been removal of some interfering substance(s) from El' by this step of purification. The concawlvalin A-dnatc of the A S F activity was resolved by DEAE-cegulase chromatography into two distinct peaks, 1 and H. ASF-I was the major peak, which mpresmts apprua_ 75% o f the epididymal plasma ASF activity. After this step of purBication, ASF-I was purified approx. 2000-fold, as opposed to ASF-II, which was p~rified only 46-fold. This study reports the pmifieation and c.harac~tlzation of the major anti=sticking factor, ASF-L White evaluating the parity of ASF-! obtained after DEAE-cehilose chromatosraphy under non-chmatming conditions by 7% polyaerylamide ~ gel eleclrophoreit was observed that A S F cons~ts of a prorein band having an Rf valttc of 0.66 and ~ c~Itraminating TABLE I P~wifico.lionofASF-I l~n epiO~-r~ol Fr~ction

S~terose-density ~"a~ent ultracentrifugation- Linear sucrose density gradients (5-22%) were prepared in Beckman ultsace~trifuge tubes of capacity 5 mL 0.1 ml of the preparation of z~I-ASF-I (apprux. 25 n8 protein; 25 000 ¢pm) containing 2 mg of ovalbumin as the marker protein was layered on t~p of 4.6 ml of the sucrose gradient. The tubes were then centrifuged at 200000 × g for 10 h at 5 ° C in a Beckman SW 50.1 rotor. After the run, the bottom of the tube was punctured with a needle end 10-drup fractions (about 0.15 ml) were collected.

El~idymal ~ Con A - ~ e|uate DEAE-ce~du|o~ chromatog~phy

Total Total Specific a~vity protein ~ v i t y (U-10 -~) (rag) (U/w~t~crein-10-~)

Punficat~m (-foM)

323

4629.6

O07

|

46g

39g.6

1.2

17

ASF-I 376 ASF-II 105 AcryIamide-Sephadex

32.5

144.6 3.23

2066

0.81 398.0

5~5

2.6

46

get e l ~ r ~ s

ASF-1

32~

117 proteins close to the orion (l~g, IB). No detectable protein bands were noted in the vicinity of the prot6n band thai migrated well through the gel Analysis o f the gel fractions for ASF activity showed that there is only one peak of activity that en~igmted with ~ protein band of R r value 0.66. Since it b very diff~ult to effete • e activity from the p o ] ~ ~ it was decided to develop a novel electrophore~ system co,talning both polyacrylamlde and Sephad~ G-200 as the matrix (Fig. IA) for the ~ of ASF.I. The ~ o phoreti~ cond~fions were manipulated to permit migrant,'on of the ASF into the Sepbad~ layer, so that the protein could he elated nearly quantitatively from tltis phase. ASF-I was purified approL 56flO-fold (Table 1) and the purified ~actor, p n ~ r v e d in 5 mM potassium phosphele (pH 7.0)/40~ glycerol/l mM PMSF. was fotmd to be stable for nt lea~t 1 month. The new technique of the g d electrophoresis system is simple and it is likely to be particularly suitable for the purification of proteins to homogeneity ff the required protein band in the polyncrylamide gel is well separated from the comaminating proteins.The l x ~ i fio~a of the Sephadex G-200 in the gel mhe can be altered so that the required protein band is d e ~ r o phorased into the Sephadex layer. The simplicity amt resolvin~ capacity (for pro~h~, DNA. RNA. et~.)

om

zt~

2.5

GEL-FRACTIONS

t

ma

I

PROTE¢~I BAND

__.~'~W///////////./~

i

5"TACKa~G

Fig_ t. G~) ~

ol,e

PROTEIN BAND

RESOLVING GEL

'

~..~

FRGrlT

: ~//////~[A)

I

FtESOLVING GEL

SEPHADEX G - ~ D

of the p ~ y a , = , ~

a.O

(B) ID~lp'a=~2a~ n:preseat,~,~n ~ the I~y'~ty~ek~ gel ~,u.roFbo,n:~tc pme~ of ASF-I ,obtained ariel the D ~ cbro~ uep of the pmifi,::ae,~ ~ , : r the . o a ~ ¢oad~dOlL After e ~ oee j~.4'wa~~ with C.oo~.~, I ~ for 1hedem~o~ of L~0~n hands and anotherSel ,,v'~.~ctio~d wltb

a Gi~o~ setom~ic gel sl~e~, the tb2dmessof each ge~ d~e being l ram. For d~ elm~n et ASF acfivhyeaghf,d sloe was Ohpe~d in 0_~ nd RPS tngdiumovernightat 6°C. An afiquot t~f the supernatant Ilmd(100 FI) from ca~ ge,I ~ was uu:d ~oc ~ a l c .ASp a~fivity under the staadasdassayce~tlo~.

66K-~

Fig. 2. SDS-polya~lamide gel cl~trophoretic pattern of ASF-I. I ~ c g s o g h o r e ~ was crag'led out accordin5 to the method of Laemmfi [4] ~ redtt~cg ¢oladitioms in the ~ of 2-mercaploeth~ol.

Rightl a ~ ASF-I;left lane: markerproteins ~ bovineserum albumin ~SA: 66000). mmlbumin(45000) and myoglobin (17000). characteristic of polyaerylamide gels and the potential for quantitative elufiou of the macromolecules f~om the Sephadex layer may make this method extremely useful for the purification of a variety of macromolecales, including proteins.

Properties of A S F PurilF. The isolated ASF-I showed the presertce of a single in'oteia band when subjected to polyacrylamlde gel electmphoresis under non-denaturing conditions and the antiosfieking activity was localized in the protein band (data not shown). A single protein band was also observed upon SDSogel elactrophoresis of the factor (Fig. 2). The data show that ASF-I has been purified to apparent homogeneity. Physical propertY. The molecular mass of the purified ASF-I was estimated by Sephadex G-100 chrom a ~ , ' a p h y [9[ to he 47 k D a (data not shown), Only one peak of anti-sticking activity was found when ASF-I was sabjected to gel filtration on Sephadex G-100, Using sucrose-gradient ultracentrifugation, with ovalburain as the marker, the sedimentation coefficient of the factor was found to he 4.25 S. The Stokes radius, a, of ASF-I, as calc,alated according to the formula. M = 4~Na3/3V [10] (where M is the molecular weight, N, Avogadro's number and V the partial specific volume) was found to be 24.1 ?,. The frictional ratio as calculated azeording to the formula: f / f o = a / ( 3 M V / 4~Na) t/3 [11] (where f/fo is the frictional ratio) was found to be 1.0. Upon SDS-gel eiectrophoresis, a single protein band was observed, with an apparent molecular

118 I

p

g3

g

.

TABLEII

"0

ASF-I { ~ at) ~ pn~m~d m~l~spe~7~ ~ ~" ~ l l l y g ~ l ~ ba a total voLo~[.~l FI ¢~[Fd~ m ~ l ~ a~ $'/~C ~¢ 2 h. AIh~ri~tdmtio~, tl~ m ~ t ~ ha~l~dag ~ ° C f~g $ ~ ~o d~uoy th~ l~J~sklasea~,~t~ a~d t ~ ~ ~ tl~a aMa,~l f~ ami-s~i~.~ ae~i,6tytm~er ~s~lard m~ay ~,,~g,.~ TSe S I , ~ idles had ao ami.s~kki~l~_,,~y ~" da~r ome.

0-1) O.4

o.8

,I~

,!6

mass of ~6 kD~ (F/g. 2). T h e values of the molk~:ular mass of ASF-I, determined by Sel~had~ get filtration and SDS-gel e: ~ophonmis methods, we~ hearty identical, indicatin~ ~a~ASF-I is a monome~ of aFPrm- 47 Id>a. Anti-sticking :aracteri.stics. Obsez'valion of the under a micr~- pe during flushing of d,,e counting chamber ~ t ~ ?S medium has shown that the acrosomal are~ the sperm-head sticks to the glass surface. Sperm~ ~ a lost their ~ l - s t ~ ; n ~ ability completely when pr~ zcated with 0.1~ Triton X-I~O, ind_',cating that 1he ce] ~stidking a d h ~ ' , e prmeins are lc~ated on the external eli sta-faoe. As shown i~, Fig. 3, the antl-s6cking activity of ASF-I iuoumse~ propoctionately up to atrprox. 4 U of the factor ('Le., oprox- 10 n g / m l or 0.25 nM AsF-I). ASF-I showe~ s ~ t ammmt of anti-sficldng activity at a c -~.ntfation as low as 0.1 riM. The spedtlc activit5 of the purified ASF-I is as high as approx. 800 U / g protein. At a saturating comamtration of 1 n M A F-I/nhibited r~arly 50--60% of sperm adhesion to gla~ There was little loss of ~ t y .when the factor was h ~e,dat 8 0 " C for 5 mira However, heat treatment at 10C C for 5 mha caased nearly 20~ loss of ASF-I activity (c-ata not shown). The activity c f ASF-I was lost completely when the factor (3.6 p g / F ] of RIPS medium) was pretreated with trypsin (25 ~tg ~ ) at 3 7 ° C for 30 rain (data not shown). The e f ~ of treatments of ASF with various glyca~sidaseshaz ~ shown in Table H. The factor was inactivated nea~ : c~mpletely when tr~tod with 1-fucosidase, a-m: ~nosidase or/t-N-acetylglu¢osaminid~s~. N~Ltramini~ ~ (20 U / r o b or ~-galactosida~ (200 U/roD at relati~ ~y high concentratiees showed oaly a slight inhibitory effect (20-30~) on ASF-L The data show that both t~e protein and sugar parts of ASF are essential for its ~ i - s t i ~ activity.

32

A S F + n ~ u i m d ~ t ~ U/rid) ASF÷ ,0-ga]z~o~=~ (200Ulna)

2.1 25

ASF~-~ . - ~

0

{0.25U/ad)

Several purified pmte~ns were evala~ed for anfi.~ acfvity with a view to find o m ~ e ~ specificity for the action o f ASF-I (Fig. 4). C a s e ~ phos'vitin, macin ami o v a l b u m ~ over a coac~mtral/am range 1-100 p.M, did not hac¢ any ~ . ~ , ~ a i f l ¢ antisticking activity. ~ , Imviae s~-.~a ~ , ~ _ #~.os~m~ retain and m y ~ ~mm~d s i ~ m c a m a~o~mts of ~ t a~ti-st/dfing a ~ , the maximal activity being ob~erv~ at l~ro~n o~nce~tratic~s as 16gh as approx. 10 p M for the first three p~meins or 1~0 FM for the last o ~ . These l m ~ i n s had Httle anti-sticking activity at c ~ u ; e a W ~ below 1 /~M. At saturating ~ t ~ t i o n s , .ASF-I, ~i~ and retain h ~ b l t ~ d ~ e r m ~ h c ~ m m ~ m by m~xly 50-~0~, whereas albumin and m y ~ m ~ i n mmt~l it by approx. 80~. ASF-I, which w ~ ma~mall~ a~ive at a conomtration as low as 1 riM, showed pfog¢~ sp~-~lcity at least 1000[~tilff~¢$ggcal¢l- ~ t]~ other p r o t d ~ tested.

e ~ 7 LOG pnonE~ c o ~ l ~ )

~

F'~ 4. ~ of ASF-I. S ~ t l ~ p,~'ifi~l lpmtc~s w~-~ ¢valuamt t'~ thor a n S - ~ a~. T[~ dam ~ am Ibe avenq~ -t,-S,D. foe fore-e~rlm~.s. @.A.~F-I; o, I~A; at f©tcln;0, g-lactotlobul~ ~ my~ol~; *., matin¢¢phosvifia.

119 ,.e,~a~ies on the ~ of ~ o~fAgF-I It is p o t m ] ~ that ASF-! may show its anti-sticking

mi-.~ty by matias tbe ~ u ~ t o ~ m t e r ceemiag d,-,nbet. To t¢~ this poui'bility, the ¢ o a t t i ~ d l a m b ~ was first incubated with a satorating concantratio~ of A,~F-I in RPS ~ (approx. 100 n$/ml) forS ~ u at r o c a mmimmtum and tlm~ fluamd with an exce~ of the buffer to remove the free factor. This pretreattmmt of the chamlmr had no eltect on tha ~ of sptmm s t i d d ~ to gLus (data not ~ n ) . ~ g that t ~ ac~fi~y of d g f a t ~ r w ~ not due to iis conting tl~ glare taufaee of the cmmtlng ~ . Treatment of intact spermatozoa (100-106 cei~/ng) with m i l a n (I00 F g / m l ) at 3 7 ° C for 60 mln caused marked iahibition (f~-~O~) of ceU ~ to g ~ (data not shown), indicating that the external surface of sperm pmsesses izrmein(s) that medinte :m-h~ion of spermatozoa to glass. ASF-I ($0 ~,/ml) when treated with an ~ of whole spemum~oa (~0 • lO~/ml) in RI~ ~ at room temperatom for 10 m~a was remaved from the m e d ~ a and the a n t i - ~ ac~ty was fomtd to be associaled with the sedim~ted ce~ls (data not shinto). These results provided evidem~ to support the view that ASF-I bimh to intact spermatozoa. To analy~ the b;,,1;ng sites or receptors of ASF on tha sponn surface, ASF-! was radinindinated and tha specific t a d i n a ~ t y of ~ labelled ASF was shown to be as high as 1050 cpm/ng l~'rol¢in.The assodation of radioactivity with ASF-I w ~ amfinned by p r e c i # t ~ , ~ the labdled factor in the ~ of ovaW,,,-;- as the cartier prme~n with lOYo~ridMoroace~cacid. Tha ~ iodmatio~ step did not h a ~ any adverse effect on the strectut~ of ASF-I since heady 85% of the anfi-s~,'~ng activity was t m m m m l in the pmified t 2 ~ I . ~ factm (data not shown). Fig. 5 shows the dose-dependet~ of the binding of ~ I - A S F - I to intact spermato'm~ The

~ sc

g 2o ~2

o'.~

&

o18

,Io

cone. OFASF-ItriM) Ftg. 5. }lind~g ~ off t~I-ASF-I to intact spegn~,,t~ conctmtraions d tlm lahdled ASF were us~ ~ the standard at~,~y~ o t ~ Thg imcmtho~ the F,'~,~,'~ a m d ~ ol th~ data.

ff

t.O

2.-0

UNLABFLLEDASF-ICONC.(nM) F'~. 6. ~ ~ t of sperm-boundt~I-ASF-Iby the mtti-stiddag factor. Standard auay ¢~aditions for the t~tdi~g ot the labelled ASF to spcmamtozoawere used, ~.~pt for the specifiedaddltlons of the unlabdled ASF.

amount of ASF bound to spermatozoa increased with the con~ntratinn of the factor and the binding was saturable at approx_ 0.75 n M ASF-I. Scatchard analysis [12] of the data su88~ts that there is positive cooperalive int,~ast]on in the bindin 8 of ASF-I to the membrane reoeptors. The K d of the binding sites was appxo~ 5.5 - I0 - t t M and nearly 9 final of ASF-I bind to 1- l 0 s calls. The effect of unlabelled ASF-I on the binding of z25I-ASF to intact spermatozoa has been investigated (Wag. 6). The unlabelled ASF competed with the r a d i m ~ v e f a c ~ f for sperm binding sites and thus caused displacement of the labelled ASF bound to these cells. Displacement of t~I-ASF by the unlabelled fa~,~r was nearly complete with 100 n g / m l (2.1 riM) of ASF.L The results demonstrated that binding of ASF-I to spermatoT~ is s p e c ~ . As already reported, bivalent metal ions may play a role in sperm adhesion to glass [3]. It is possible that ASF may show its anti-sti~king activity by chelating actino. This, la3wever, appears unlikely, since MgCI 2 (1 raM) a n d / o r CaC~ 2 (1 mM) had no effect on the activity of ASF-I (data not shown). Effect o f A S F on sperm-sperm adhesion Spermatozo~ derived from certain parts of epididymis (e.g., corpus) have b e e , shown to undergo a 881utinatinn when incubated under a defined condition at 3 7 ° C [13]. Evidence has been presented for the presence of undefmed anti-agglutinin protein(s) in EP, seminal plasma and prostatic secretion that inlfibit the agglutination of spermatozoa derived from several maln~Ali~n species [14]. We have also observed that goat EP has a high capacity to inhibit agglutinatiun of spermatozoa obtained from the distal end of goat corpus epididym~s (unpublished data). As shown in Fig. 7, goat ~'pus-epididymal spermatozoa when incubated at

120

;

.

-=

t~

.

.

~ ~

~ " ~

.

•

:•:.~.

~

[

:

)i: ?

Fi8~ 7. Inlatbition of slmrm-sp~m a l l g l ~ by ASF-L The a a ~ action of ASF-I ~ sperma~,~ w~s ~ fe~ca~ag Ihe method of Daclhe~tx¢t aL [13] v4th some mod~f~iom. ~ from the d~tal ~ regm~ (Le-, th© rqpo~ join prior to ~tada) ed"the epididyn~ win: ~aTacted in RPS nned~um, ~ m ~ at 4L~x g f ~ ~ taka at so~e~ t~mp~m~ ~ ~ ~ ~ ~ ~ ~ resuspcnded ia RPS medimat Ce~ (25- 10e/a~4i)v.~e Ihea iacttbated at 3"/°C for 50 m~a ~ ~ ~ ~ ~ d ~F-I m a ~ ~ of 0~ ml or" RPS medium. After the ~ d~e ceRs w,~e obsm'ved uader a phase-coa~ ~ ~ ~ ~ ~ spermal~0~oawith a magnillcatio~of ZTOx_ (A) C ~ t r ~ ( ~ t ASF-I~ (B) W]ffl AS~-I (4 n~ml}. (C) With ASF-I (20 ~J~I]L {D} ~ftlh ASF-I (5O aS/'~). 3 7 ° C for l h in RPS medium showed head-to-head agglutination giving rise to large d u s t e r s of cells. ASF-1 showed a high tendency to inhibit the sperm agglutination. A S F at a concentration as low as 4 n g / m l signilic a n d y inhibited this s p e r m - s p e r m adhesion as observed by the reduction in size o f the cell chistem. A S F at a concentration o f 50 n g / m l showed maximal inhibitory effect when most o f the cegs remained in free suspension. ASF-I (50 ng/ml}, when a d d e d to the prcformed sperm clusters, also showed high efficacy to dissociate the cells from the cell agglutinates (data not shown), thereby showing that it can also reverse the process o f s p e r m - s p e r m adhesion.

Distribution of anti-sticking activity. The ~ c u r ~ l c e o f A S F activity in different tissues and fluids has been investigated using goat canda-epidldymal spermatozoa as the cell model (Table ii1). Anti-sticking activity has been detected in all the tissues and fluids tested. ] ' h e specific activity of A S F was markedly higher in blood serum than in any other sources. Diseusskm Evidence has earlier been provided for the existence of a heat=stable factor in blood serum that decreases the

adhesion of human ¢rythrocytes to glass [15] a n d B H K cells to tissue culture glass o r plastic [16]. H o w c a ~ , the serum A S F has not been purified and litlle is known about its biochemical charactcsistics. Preliminary slndk s from our hiboratoD' provided evidence for the occtwvmcc in goat E P o f a potent A S F activity that sixongly

inhibitc~d adhesion of spermatozoa to glass [3]- This study reports, to our knowledge for the f~s[ time, the purification and characterization of an anti.sticking factor.

ASF-L the major anti-sticking activity of EP. has been purified to apparent homogeneity. It is a glycoprotein, as evidealced by its binding to coacanavalin A and sensitivity to the action o f several 81ycosidascs (Table II). The observation that nearly 9 0 ~ proteins o f E P that did not bind to Con A-agaruse had no delectable antislicking activity indicates that ASF-I is a highly specifm anti-sticking protein. The high potcacy o f A S F - ! at a concentration as low as 0.5 n M and inefficacy o r eflectivaness at markedly higher concentrations ( 1 - 1 0 0 p M ) o f several purified proteins (Fig. 4) strengthened the above view. T h e markedly lower anti-sticking activity o f several purified proteins, such as albumin, fetuin, ~-Iactoglobulin and myoglobuSn, as compared to A S F - L

121 TABLElit Fn~h ~

bloed wr~ ceaected ia c i u a ~ mb~ aad tlga ~ i f u l ~ d

at .f~O×g for tO tramto ol~ain t~14me ~ ~ w~e ~/tom g0at ~ md EP was i~epamd by the ~ tlmatibedia ~ and Me:beds.The ~ t , - ~ ~ mm~washed twice,a,lth RI~ ~ to remevecem~.i~t;~ EP_ The ~ were a ~ . ~ aad ~a~ed e x t e a ~ ~ Itl~ medium to t ~ v e

co~mm~

spol~'-~

~ wea as EP. W a ~ e d

slammaoze~ e f / d / d ~ i ~ aad otl~ertis~es mac ~ in a Tefltm I m ~ amd the ~ ~ere ceattifuued at ISO0~ Xg for tO role. "the ge~dti~gst~p¢:n~taatl]h~h, b~oodp6umlaand EP w=e d~yr~l c ~ a m ~ almmt geS medium,prttw to a~ay o; aeeti-stigk~gac~vityundegthe s~mdafd mlsayc~e~tiom. The data ~town a~em a ~ + g.D_n:W,.,,,.m=,~eof fo~ e~edmew_~ Sc~ce of ~SF a ~ t y

Spmf= act~ty d ASF (u/rag pmmla)

Egoodplasm~ Heart Liver Kidaey Teslls Spe,'~

2600±210

~ly,dais

v~,~

126i 60 2115+ 80 2to_+tOO 70:~ 10 29± 8 674- 10

p~z

,~± to

suggests that these proteins may serve as ~ t c anti-sticking factors. The anti-sticting activity is not characteristic of a ~ycoprotein, since most of the EP glycopmttius and purihed glycoproteins, such as ovalbumin, casein and mucin, did not show any antisticking activity (Fig. 4). The fmding that the coll-stiddng ability of the intact spermato~ou is lazgdy lost following treatment of the cells with trypsin suggests that specific ecto-prme~s) mediate attachment of cells with glass. The results are consislt~t with the view that ASF-i binds specifically to the outer surface protein(s) (n~ptors) and thereby destroys their ~t~lh,-~vecharacteristics. This view is sopported by the ~ o n that 12SI-ASF-I binds to intact spermamzou with high affinity and that the binding is saturable and displaceable with the tml~helled factog. Rapid uptake of the unlahelled ASF-I by intact spennatazoa with concomitant marked loss of their cell-sticking ability and the fmding that ASF did not show its activity by coating the glass surface strengthened the above notion. The observation that the dose-dependence of ASF-I for its anti-sticking activity (Fig. 3) closdy resembles that for its binding to intact cells (Fig. 5), indicates a close cogrelation between the Y-$F-receptor interaction and its anti*sticking activity. Both immature and mature epididymal spermatozoa showed high affinity to bind to the glass surface of the haemocytometer counting chamber ]2,3], which is extensively used fog n g motility subjectively as well as objectively ~ i n g v ~ sophis"ticated photogr~hic

tca~aiqn~ 11], As mentioned above, sperm adh~ion to the counting chamber may pose a serious artifact fog sperm motility assays ]2,3]. It is thus likely that the earlier studies on motility assays, with special reference to those on the occurrence of various motility initiating, promosing and maintaining factors [17-26], may be complicated by artifacts because of the problems of ceg adhesion. Since ASF-I is a specific and potent inhibitor of sperm adhesion to glass, it is likely to he ideal for improving markedly the existing motility assays by diminating the possibility of cell-sticking artifacts. Cell-cell adhesion is believed to play a vital role in cellular fogalation [27]. Whole cells also interact with a variety of surfaces (substratum) of biological or nonbiological origin, such as connective tissue elements, glass, plastic, etc. [27]. It is of interest to note that the m~hanis~m of call-substratum interaction closely resembles that of the cell-cell adhesion [28]. Several cellsurface adhesive proteins (e.g., fibronectin, aadherins, etc.) that may mediate these adhesions have been identified ]29,33]. However, little is known about any specific protdn that m y regulate these interactions. Occ~rreuce of ASF activity in a variety of tissues (Table 111) suggests that ASF may have a biological role not restricted only to spermatozoa. At present, little is known about the physiological significance of ASF. The sperm-glass adhesion phenomenon is an exmaple of ceH-substratom adhesion [28] and the finding that ASF is also a potent anti-agglutinin fog sperm-sperm adhesion (Fig- 7) suggests that ASF m y have an important role in the regulation of mammalian cell-cell and cell-substratum adhesion by serving as a specific and potent anti-agglutinin. Physiological fluids containing free suspens~ns of cells are thus expected to have relatively much higher specific activities of ASF than the tissues. The observation that the specific activity of ASF in blood plasma that contains several types of cell (erythrocytes, leukocyses, etcJ in free suspension is markedly higher than in the tissues tested (Table III) is consistent with the above view. Spermatozoa in certain human semen samples have been reported to be present in agglutinated form and having low fertility [34]- Although earlier investigators demonstrated in the male reproductive fluids the presence of anti-agglutinin(s) that inhibited epididymal spenn-sperm adhesion, little is known about the identity of these molecules [13,14]. This study shows that ASF represents at least one of these sperm anti-agglutinins. Inefficacy of ASF to cause complete inhibition of sperm-sperm adhesion (Fig. 7), is perhaps related to its ability to inhibit 50-60~ spermatozoa adhesion to glass. It thus appears likely that ASF may f,crv¢ as a potent anti-asglutinin, thereby inhibiting sperm-sperm adhesion and the adhesion of spermatozoa to the cells of male and female reproductive tracts. This postulated role of ASF will permit sperma-

122

t o z o a to r e m a i n i n tim free s t a t e necessm'y for f o ~ v a r d m o t i l i t y a n d fertility a n d i t s deficiency will c a us e infertility b y a g g l u t i n a t i n g the cells.

Ac~m,~eme~ A research fellowship offered t o N . R . b y the C o u n c i l o f Sciendl'w a n d I n d u s t r i a l Research, N e w ~ is gratefully acknowledged. T h e a u t h o r s are gra t e ful t o D r . S.C. Pak r ash i for h i s interest i n this study. T h e a u t h o r s also ack n o w led g e the technical a s s i s l a ~ e o f M i s s K . C h a l a ' a b o n y a n d M r s . S. Banerjee fc~ the preparation of epididymal plasma.

13 Dacheu~ J.L, I ~ l e i ~ m . M. and ~ Y. 11~3} J. FeniL 67, IBl-t89. 14 ~ P.E., Ktlg~ram,/.E- taxi M ~ Ph- (19e~ Fer6L S~nt3L$, 241-255. 15 G ~ l ~ . , J.N. Wa:d, ltdL a~l RI~IL,C.F. {1971) J, C d I~I~i~L T?, 16 R o ~ u a ~ S.. R ~ . W~ WaRher, B, (]6emm. IL a~l Un~reu, J. (1974i) Methods EIOZ3~L 32, ~f1-609. 17 ~ T.S. and ~ D.D. 097g) J. I ~ t Chem. 253, 6744-6750. i s Chaud~ry. D-P. ~ ~. ~r ~Mr . G.C O ~ ~ 15. a~-497. 19 G.ma-, R.D. aml Talwax, G.P. (197.~* Int $. Fe~]. 20,133-1~. 20 HofJdms, D.D, BrazIL EL and Aco~t, T ~ f197~ Fed- IPm~ 3"/, 2534-2.~47.. 21 Mmiea., Z. aad C]ha~t~ M.C. 0.971) J. ~ Femli ?J~ 7K/-254. 22 Mc~-Ion, B. and Cha~, T~S,K. O9"13)J'. Retmrod. Femtx'L 35,

Referemoes 1 Am~lr, R.D, Dubin. L ~ 1 ~cJhmcnl'~d~C. ( 1 ~ ) Ferl~ SI~-I'L 34,19"/-215. 2 Stepl~as, D.T~ Actor, T.S. and Hos]kins~ D.D. (1981) Biol. Reprod. 25, 945-949. 3 Roy. N . Majumd~. G.C and ~ CJK. (1~5) Androkgia 17, 200-2~6,. 4 ~ U.K~ (1970) Nature (London) 227, 6~9-695. 5 Mm'kwe~ M-A-K- 09~2) Ang- Biochem- 12~, 427-43Z 6 Rey, N. and Majumdet. G.C. (1~86) I~p. ~ ~ 164, 415-425. 7 Low~. OH., ~ m e g h , N J . Farr. AJ - ~ Randall RJ. {1951) .If.Biol. Chem. 193. 265-275. 8 Bensadm~ A. amJ Weismein, D. O976) Anal. Bioche~- 70, 241-250. 9 Whlt~er. J.R. (1963) Anal Chem. 35,1950-1953. tO Acke~s, G.K~ (1964) B~..be~f,u'y 3, "72~7~0. 11 Siegal. LM. and Monty. KJ. (19~6) ~ Biophy~ ~ 112. 346-362. ]2 Ginsbers, B.tl. ~ CP-,. and R o ~ J. 0976) Bioch~ ~ y s . Act~ 4o,3, 227-242.

32,107-114. 2# Mul~r, B. a~l @ , C. (1978) J. R q ~ d . Flnt1~ 54.16?-r/2. 2.$ S~eth, A.R.. G u a j ~ r , A.N. and 5 ~ h . G.V~ flggl) A~m~g~a 13, 14~.-1,46. 26 Voglma)T, l.[~- am:l Wl~t~ LG. O979) ] ~ . I~]~ad. 20, 2~&..293. 27 H u ~ , R.C~ Peu, S.DJ. and V'~:be~, P. 09~0} ~ ~ ~ aml Motitity. The ~1S,jmp~mem d the l h ~ .Y,a o ~ far ¢~i Ie~lagy (Gmiz, .~S.G. and Pats. J.D~ edr~), pp. 3"z~356, Cam. brk~e U m ~ d ~ y ~ ~ U.K. 28 ~rarm,d. DJL (19B6) J. C.¢~ S~L S ~ L 4. 221-237. 29 Dam.~-y. C.H.. Ricba. J~ Sml~t. D~ Kaudsm, I ~ real l~e~.. C.A. (lq~3) Cell 34. 455-466. 30 F . d ~ n ; O.ML (1984) Exp. Cell Res. 161,1-16. 31 Nine, A. and T ~ e i ~ i , M. (19'86) J. Ce~ B~I. 103, 2649-26~. 32 Rol~t~, G.P, a~l Brunt, J. 0985) I~ochem. J. ~ 67-70. 33 y ~ m ~ ICM. and ~ K. (1978) N m (L~u~-) 275,179--185. 34 ~

A. a~d ~

,

D. (19~3) ~ . J. UroL 45, 559~562-