Quantitative Electrophoretic Study of the Modification of Sperm Plasma Membrane by the Ampullary Gland in the Golden Hamster

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1 Archives of Andrology Journl of Reproductive Systems ISSN: (Print) (Online) Journl homepge: Quntittive Electrophoretic Study of the Modifiction of Sperm Plsm Membrne by the Ampullry Glnd in the Golden Hmster P. H. Chow, A. C. Y. Yuen & L. Y. L. Cheng To cite this rticle: P. H. Chow, A. C. Y. Yuen & L. Y. L. Cheng (1995) Quntittive Electrophoretic Study of the Modifiction of Sperm Plsm Membrne by the Ampullry Glnd in the Golden Hmster, Archives of Andrology, 34:2, 53-61, DOI: 1.319/ To link to this rticle: Published online: 9 Jul 29. Submit your rticle to this journl Article views: 3 View relted rticles Citing rticles: 1 View citing rticles Full Terms & Conditions of ccess nd use cn be found t Downlod by: [ ] Dte: 9 Jnury 218, At: 16:31

2 QUANTITATIVE ELECTROPHORETIC STUDY OF THE MODIFICATION OF SPERM PLASMA MEMBRANE BY THE AMPULLARY GLAND IN THE GOLDEN HAMSTER P. H. CHO Deprtment of Antomy, Fculty of Medicine, The Chinese University of Hong Kong, Hong Kong Downloded by [ ] t 16:31 9 Jnury 218 Keywords A. C. Y. YUEN Deprtment of Antomy, Fculty of Medicine, University of Hong Kong, Hong Kong L. Y. L. CHENG Deprtment of Biochemistry, Fculty of Medicine, University of Hong Kong, Hong Kong Plsm membrne proteins were extrcted either from epididyml sperm fter incubtion with mpullry glnd secretion or from uterine sperm derived from surgiclly treted mles belonging to the following groups: TX, excision of ll ccessory sex glnds (ASG); AGX, bilterl excision of mpullry glnds; AG, excision of ll ASG except mpullry glnds; nd SH, shm-operted. Totl membrne protein, glycoprotein, nd SDS-PAGE of individul polypeptide subunits were quntified. After incubtion with mpullry glnd secretion, both protein nd glycoprotein concentrtions of epididyml sperm membrne were incresed. The protein profile ws lso significntly ltered, with the removl of the 43- nd 71-kD subunits nd the ddition of the 36- nd 5-kD subunits. The in vitro results confirmed this proteolytic effect of mpullry glnd nd other ASG on the 43- nd 71- kd subunits, despite reduction in membrne protein concentrtion. Modifiction of the 17-, 2-, 25-, 28-, 56-, nd 66-kD proteins were lso observed. This report is the first demonstrtion tht the mpullry glnd is cpble of modifying proteins on the sperm surfce. mpullry glnd, spermtozo, plsm membrne, SDS-PAGE, hmster, ccessory sex glnds, electrophoresis protein, glycoprotein, This reserch ws supported by University of Hong Kong Ermrk Reserch Grnt 337/31/7. Address correspondence to Dr. P. H. Chow, Deprtment of Antomy, The Chinese University of Hong Kong, Shtin, New Territories, Hong Kong. ARCHIVES OF ANDROLOGY 34:53-61 (1995) Copyright 1995 Tylor & Frncis /95 $1. +.OO 53

3 54 P. H. Chow et l. Downloded by [ ] t 16:31 9 Jnury 218 The mpullry glnds of mmmls re glndulr enlrgements rising from the mpulle of the vs deferenti. They my ttin very lrge size s in the Equide or be vestigel s in the guine pig, bor, nd mouse. In some rodents, such s rts nd hmsters, they form four discrete glndulr enlrgements. Informtion on mpullry glnd secretion from vrious mmmlin species is frgmentry. This secretion contins ergothionine, glycerylphosphorylcholine [9], cid phosphtes, nd citric cid [2]. Hydrolyses hve been demonstrted in the glndulr distl vs deferens of humn [8], nd glycosidses re present in stllion mpullry secretions [6]. In the golden hmster, mpullry glnd secretion is wtery fluid contining 1 mg proteidml [2]. Removl of mpullry glnds results in higher-thn-norml rte of embryonic wstge leding to significnt reduction in fertility rte, lthough the fertilizing bility of spermtozo ppers to be unffected [4, 51. A similr surgicl tretment results in higher rte of polyspermic fertiliztion in the golden hmster [3]. At the moleculr level, the sperm plsm membrne plys significnt role in gmete interctions. Structurl nd physiologicl modifictions of spermtozo re completed in the epididymis. Little is known bout postepididyml modifiction of spermtozo. This study ws conducted to evlute postepididyml modifiction of the sperm membrne protein s relted to the mpullry glnd. MATERIALS AND METHODS Animls. Golden hmsters (Mesocricetus urtus) used in this study were rndomly bred nd mintined in diurnl cycle of 14 h light: 1 h drk. All femle hmsters were inspected dily for vginl secretion for t lest two consecutive norml cycles before mting. here necessry, nimls were scrificed by chloroform inhltion. Chemicls. All chemicls used were of the purest grdes; unless otherwise stted, they were purchsed from Sigm Co. (USA). Preprtion of Sperm Membrne Frction. Plsm membrne of sperm ws extrcted with.5% NP-4 in ice for 2 min nd centrifuged t 1,OOOg for 1 min. Electron microscopy ws used to ensure tht only the cell membrne ws removed. The structure of the sperm, including tht of the crosome, ws intct. Superntnt contining the solubilized membrne protein ws concentrted with Centricon-3 (Amicon) microconcentrtors nd its protein content ws determined in duplictes by the Lowry method [14], using bovine serum lbumin (BSA) s the stndrd. The pellet ws used for determintion of DNA. Quntittion of Sperm Surfce Glycoproteins. Sperm surfce glycoproteins were lbeled with the glctose oxidsehritited sodium borohydride method. One milliliter of wshed sperm (2x 1 O7 cells/ml) suspended in.264 mm sucrose/5 mm Hepes buffer t ph 7.4 ws incubted with 2 units of glctose oxidse (EC ) nd NB3H, (Amershm) in n Eppendorf tube t room temperture for 2 min. Spermtozo were then wshed twice, followed by plsm membrne extrction, s described in the lst section. Membrne protein ws precipitted with 2% trichlorocetic cid (TCA) nd.1 mg/ml BSA. The precipitted protein ws collected onto glss microfiber filter (GF/F, pore size.7 mm), which ws then wshed twice with TCA nd ir-dried for 3 min before being plced into scintilltion vil contining phse combining system (PCS, Amershm). After 3 min, the rdioctivity of the smple ws counted by scintilltion counter (Beckmn, LS581) for 1 min. Extrction nd Quntittion of DNA From Sperm Pellets. It ws considered more ccurte to use totl DNA s n index of the quntity of spermtozo used for membrne extrction insted of sperm

4 Ampullry Glnd nd Sperm Plsm Membrne 55 count. The sperm pellet from membrne extrction ws resuspended in 4.5 ml of NE buffer mde up of.75 M NCl nd.24 M EDTA. Then 25 pl of 1% sodium docecyl sulfte (SDS), 75 ml proteinse K (2 mg/ml), nd 15 pl H,O were dded nd incubted overnight t 5T. After centrifugtion t 3g for 1 min, DNA from the superntnt ws extrcted twice with 5 ml phenol (phenol: CHCL, = l:l), fter which the dissolved phenol in the DNA solution ws removed with CHC1,:isomyllcohol (24:l). DNA in solution ws precipitted with.2 M NCl nd 8.2 ml cold (-2 C) 1% ethnol nd vcuum-dried. DNA ws then dissolved in 1 ml Tris-HCI, 1 mm EDTA buffer t ph8.. Absorbnce t 26 nm ws mesured. An A,,, of 1.O indicted 5 mg/ml double-strnded DNA. Sensitivity of the ssy ws *.5 mg/ml double-strnded DNA [16]. All ssys were done in duplictes. Downloded by [ ] t 16:31 9 Jnury 218 SDS-PAGE nd Quntittion of Sperm Membrne Proteins. A 1.5 pl membrne smple with protein concentrtion of pproximtely 1 mg/ml ws used for ech nlysis. The protein ws dissocited with 2.5% SDS, 5% p-mercptoethnol nd heted to 1 C. SDS-PAGE ws performed on the Phstsystem (Phrmci) using PhstGels (Phrmci) of grdient 8-25%. The running condition ws set t 25 V, 1 ma, nd 15 C for 8 Vh. Low moleculr weight stndrd (Phrmci) ws used. Polypeptide bnds were stined with Coomssie brillint blue (sensitivity: 2-3 ng protein per bnd, mnufcturer s specifiction; Phrmci) nd preserved with 15% glycerol. The bnd intensity ws determined by Ultroscn XL lser densitometer (Phrmci). The re occupied by ech pek of the intensity profile ws mesured with n utomtic imge nlyzer, VIDAS (Kontron Imge Anlysis Division). To compre bnd intensities from different smples, polypeptide subunit intensity index ws devised, which ws rtio of the reltive re occupied by ech pek (bnd) to protein concentrtion of the smple. Interction of Spermtozo ith Ampullry Glnd Secretion in Vitro. ith the id of dissecting microscope, mpullry glnds were removed nd clered of connective tissues nd blood. Severl cuts were mde on the tissue with pir of fine, shrp scissors to relese the secretions, which were then spirted with microcpilry tubes. Cud epididymides nd the ttched vs deferenti were removed nd clered of blood nd connective tissues. A smll cut ws mde on the epididymis to llow collection of spermtozo by retrogrde perfusion through the vs deferens with Dulbecco slt solution without BSA. Spermtozo were wshed twice by swirling them gently in Petri dish. The finl concentrtion ws djusted to 5 x 17/mL. Two-milliliter liquots of sperm suspension were dispensed into 3-cm culture dish to which 3 pl (1:I diluted, contining 5 mg/ml protein) of the mpullry glnd secretion ws dded. About 3-5 pl secretion ws collected from one niml. Incubtion ws crried out in 95%, nd 5% CO, t 37 C for 45 min. Insted of mpullry glnd secretion, 3 pl of sline ws dded to the control incubtion. Sline, rther thn protein, ws used s control becuse the im ws to study interction of ll proteinteous components of the mpullry glnd secretion with epididyml sperm. After incubtion, sperm seprted from the incubtion medium by centrifugtion t 5g t 4 C for 5 min. The pellet ws then wshed twice by swirling gently in Petri dish with 2 of ml of 5 mm Hepes/.246 mm sucrose buffer t ph 7.4 contining 2 mm phenylmethylsulfonyl fluoride (PMSF) nd 1 mm iodocetmide followed by centrifugtion. The plsm membrne protein ws extrcted s described bove. Membrne protein ws then dilyzed in double-distilled wter, concentrted with Centricon-3 (Amicon), nd stored t -7 C. SDS-PAGE nd quntittion of the smples were crried out. Interction of Spermtozo ith Ampullry Glnds in Vitro. Vrious ccessory sex glnds were removed from 7-week-old mle hmsters s described by Chow et l. [5] to give the following four experimentl groups: SH, shm operted nimls; AGX, bilterl excision of the mpullry glnds; AG, bilterl excision of seminl vesicles, ventrl prosttes, dorsolterl prosttes, cogulting glnds; nd TX, bilterl excision of ll ccessory sex glnds. Sperm ws flushed out with 5 mm Hepesl.264 mm sucrose buffer, ph 7.4, contining 2 mm phenylmethylsulfonyl fluoride (PMSF) nd 1 mm iodocetmide in phosphte-buffered sline from the uterine horns of 8 to 12-week-old femle hmsters 3 min fter

5 56 P. H. Chow et l. mting with mles from the vrious tretment groups. Spermtozo were pooled, wshed twice by swirling the petri dish gently, nd then centrifuged t 4 C for 5 min in round-bottomed test tubes. Depending on the tretment group, ech sperm smple might represent collection from 2 4 mtings. The finl sperm concentrtion ws djusted to 2 x lo7 sperm/ml for immedite extrction of plsm membrne. Sttisticl Anlyses. Dt ws nlyzed with nlysis of vrince (ANOVA), followed by twosmple Student t test. Significnce ws determined t the 95% nd 99% levels. Downloded by [ ] t 16:31 9 Jnury 218 RESULTS Interction of Spermtozo ith Ampullry Glnd Secretion in Vitro Both protein nd glycoprotein concentrtions of epididyml sperm were incresed fter incubtion with mpullry glnd secretions (Tble 1). Incubtion with mpullry glnd secretions resulted in the removl of the 43- nd 71-kD subunits nd the ddition of the 36- nd 5- kd subunits (Figure 1). Moreover, the reltive quntities of proteins in the 25- nd 66-kD subunits of the control were significntly decresed, but those of the 17- nd 28-kD subunits were incresed. Interction of Sperm Plsm Membrne ith Ampullry Glnd Secretions in Vivo A prllel in vivo study ws crried out by compring uterine spermtozo recovered from femles mted with TX (the control, with totl bltion of ccessory sex glnds) nd AG (mpullry glnds only) mles. Protein nd glycoprotein content of the AG sperm ws significntly less thn tht of the TX sperm (Tble 2). SDS-PAGE nlysis showed tht the 43- nd 71-kD sperm membrne protein subunits of TX mles were not found in the AG group, but new 23-kD subunit ppered. Quntittively, the 14-, 25- nd 66-kD subunits of the AG group were significntly reduced, but those of the 17-, 28-, 36-, nd 41-kD polypeptide subunits were incresed (Figure 2). The effect of the bsence of secretion of the mpullry glnd on uterine sperm plsm membrne ws studied in mles whose mpullry glnds hd been removed (AGX); shmoperted (SH) mles were controls. Although not sttisticlly significnt, the glycoprotein content of the AGX sperm ws slightly decresed (Tble 2). SDS-PAGE resolved 13 polypeptide subunits in both groups (Figure 3). Removl of the mpullry glnd resulted in the dispper- TABLE 1 Protein nd Glycoprotein (Lbeled with NB3H, nd Glctose Oxidse) of Plsm Membrne from Epididyml Spermtozo Incubted with Ampullry Glnd Secretion Protein Concentrtion Glycoprotein (DPM) Incubtion Type N (w4ml) Sperm DNA (w)-' Sperm DNA (pg)-' Sline (control) 4 49 f * 4 Ampullry glnd 4 56 * 3* 156 f 8* Nore. Vlues re mens * SEM. N = number of incubtions. *p I.5 compred with control.

6 Ampullry Glnd nd Sperm Plsm Membrne Downloded by [ ] t 16:31 9 Jnury 218 x z 3 >- k 25 cn Z w I- z 2 t Z cn w!i 1 >- 1 5 = AG secretion incubted (7) MOLECULAR EIGHTS (KD) FIGURE 1 Epididyml sperm plsm membrne protein fter incubtion with mpullry glnd secretion. The membrne protein ws nlyzed with SDS-PAGE nd the reltive quntity of protein in ech subunit ws expressed s polypeptide subunit intensity index (see results section). AG, mpullry glnd. TABLE 2 Protein nd Glycoprotein (Lbeled with NB3H, nd Glctose Oxidse) Concentrtions in the Plsm Membrne of Uterine Spermtozo from Femles Mted with SH (Shm-Operted), TX (Abltion of ll ASG), AG (Bilterl Abltion of Cogulting Glnds, Dorsolterl Prosttes, Seminl Vesicles, nd Ventrl Prosttes), nd AGX (Bilterl Abltion of Ampullry Glnds) Protein Concentrtion Glycoprotein (DPM) Tretment Group N (PdmL) Sperm DNA (w-' Sperm DNA (pg)-' SH AGX TX AG 19 f 3 22 f 2 63 f 5 2 f 1" 6127 f f f f 7b Note. Vlues re mens f SEM. N = number of sperm smples collected from 2-6 mtings. O,*p I,1 compred with TX group.

7 58 P. H. Chow et l. 25, * Downloded by [ ] t 16:31 9 Jnury 218 s * * :ii MOLECULAR EIGHTS (KD) FIGURE 2 Plsm membrne protein of uterine sperm from TX (totl bilterl excision of ll ccessory sex glnds) mles nd AG (bilterl excision of cogulting glnds, dorsolterl prosttes, seminl vesicles, nd ventrl prostte glnds) mles. Membrne protein ws nlyzed with SDS-PAGE, nd the reltive quntity of protein in ech subunit ws expressed s polypeptide subunit intensity index. nce of 28-kD subunit, while quntittively the 14-, 2-, 23-, 32-, 41-, 56-, nd 66-kD subunits were ll intensified. DISCUSSION The effect of mpullry glnd secretion on sperm plsm membrne ws studied both in vitro nd in vivo. The in vivo study ws complicted by the presence of uterine secretions. The pprently prdoxicl differences in the quntities of membrne proteins in the two studies reflects the complexity of interctions of sperm membrne, uterine secretions, nd ccessory sex glnd secretions. In the in vitro system, mpullry glnd secretion increses the mounts of protein nd glycoprotein in the plsm membrne of epididyml spermtozo; but when the sperm is exposed to uterine secretions s well, s in the TX vs. AG groups, mpullry glnd secretion reduces membrne protein concentrtion. Compred with the shm-operted controls, removl of the mpullry glnds (AGX) hd no effect on the quntities of protein nd glycoprotein in sperm membrne. Thus observtions mde in vitro my not ccurtely reflect the outcome of complex interctions tht tke plce in functionl situtions. Sline incubtion of epididyml spermtozo lone cuses chnges in their plsm membrne protein profile. The 43- nd 71-kD subunits were bsent while the 36- nd 5-kD sub *

8 Ampullry Glnd nd Sperm Plsm Membrne 59 Downloded by [ ] t 16:31 9 Jnury 218 units were present in spermtozo tken fresh from the cud epididymis (unpublished observtions). Chnges fter sline incubtion presumbly re crried out by ectoproteses intrinsic to the sperm. Incubtion of epididyml sperm with mpullry glnd secretion leds to the disppernce of the 43- nd 71-kD subunits. It is probbly the result of protese ctivities present in mpullry glnd secretions. The in vivo study confirmed this ssumption. Both sub-units were present in the TX group but disppered in the AG group. They were lso bsent in the SH group, which hs ll the ASG intct. However, removl of the mpullry glnd only in the AGX group lso resulted in the disppernce of the 43- nd 71 -kd subunits, indicting tht other ccessory sex glnd secretions my exert the sme effect s tht of the mpullry glnd. ilson et l. [2] demonstrted proteinse ctivity in secretions of the prosttic complex of the rt. The 17-, 2-, 28-, 56- nd 66-kD subunits pper to be modified minly by secretion from the mpullry glnd. The 28-kD subunit, present in both the TX nd SH sperm, is bsent in AGX sperm. Its presence is enhnced upon contct with mpullry glnd secretion under both in vivo nd in vitro conditions. It would pper mpullry glnd secretion protects the removl of this subunit by the other ASG. The 17-kD subunit is enhnced by mpullry glnd secretions under both in vivo nd in vitro conditions. The rest of the peptides in this group pper to be intensified by coting proteins produced by the other ccessory sex glnds (ventrl prostte, seminl vesicle, dorsolterl prostte, nd cogulting glnds), but re reduced by mpullry glnd secretion, They include the 2-, 56-, nd 66-kD subunits. Spermtozo re 35, X D z 2- t v) z I- z t Z 3 m 3 v, b- n I * * SH (7) = AGX (5) * T * T MOLECULAR EIGHTS (KD) FIGURE 3 Plsm membrne protein of uterine sperms from AGX (totl bilterl excision of mpullry glnds) mles nd SH (shm-operted) mles. Membrne protein ws nlyzed with SDS- PAGE nd the reltive quntity of protein in ech subunit ws expressed s polypeptide subunit intensity index.

9 6 P. H. Chow et l. Downloded by [ ] t 16:31 9 Jnury 218 coted with wide vriety of mcromolecules upon exposure to seminl plsm [21]. Specificlly, proteins from seminl vesicles nd cogulting glnd interct with the surfce of humn nd mmmlin epididyml spermtozo [ 1, 1, 15, 171. A cltrin-like seminl vesiculr protein, which binds to nd my protect spermtozo from clcium influx, hs lso been demonstrted in the guine pig [7]. In our study, evidence of interctions of uterine nd ASG secretions cn be trced in two subunits. Thus, uterine secretions pper to enhnce, while ccessory sex glnds, in prticulr the mpullry glnd, reduce the intensity of the 14-kD subunit. On the other hnd, the 23-kD subunit is seen in both profiles of sline-incubted nd mpullry glnd secretion-incubted epididyml sperm membrne, s well s those of the AG, AGX nd SH groups, but not the TX group, thus suggesting tht it is removed by uterine secretions but cn lso be formed by ASG secretions. This 23-kD subunit my represent more thn one protein. Little is known bout the physiologicl significnce of seminl proteins. An overll effort of mintining the fully functionl cud epididyml spermtozo in repressed stte until they meet the oocyte hs been proposed [ 121. Our quntittive nlyses of the uterine sperm plsm membrne seems to suggest tht the mpullry glnd my help to remove lot of these coting proteins when the spermtozo re trnsferred to the femle genitl trct. This cn be effected by the hydrolses nd proteinses present in its secretion [6]. Physiologiclly, this might simply fcilitte gmete trnsport by reducing the weight on the spermtozo or, t more complex level, expose or protect ctive surfce moieties. The 25-kD subunit is consistently present in the electrophoretic profiles of sperm plsm membrne protein from ll experimentl groups in our present study; this subunit my correspond to the puttive zon pellucid binding protein (Mr = 26,) proposed for the hmster [19], thus explining why fertiliztion is not ffected even in the bsence of mpullry glnd nd ll ASG [4]. Its intensity is significntly reduced when spermtozo re exposed to mpullry glnd secretion in vivo nd in vitro, but is not chnged when the glnds re removed (SH vs. AGX). The reltionship of this observtion to the reduced fertility observed fter removl of mpullry glnd remins to be explored. Our previous studies suggest tht two fctors my ccount for the reduced fertility in mtings involving mles without mpullry glnds: polyspermy [3] nd embryonic wstge. The bility of oocyte to block polyspermic fertiliztion is initited by components on the sperm surfce [ 1 I] while bnorml embryonic development my lso be relted to the sperm surfce s sperm membrne components fuse with tht of the vitelline membrne t fertiliztion [13]. This report is the first demonstrtion of modifictions of sperm membrne by the mpullry glnd the mmmls. Further studies re needed to llow us to elucidte the functioning mechnism of this glnd nd the prt it plys in the mintennce of fertility. REFERENCES 1. AumUller, G, Huntemnn S, Klus P (1989): Seminl proteins binding to spermtozo. Dev. Ultrstr Reprod Chow PH, Chn C, Cheng LYL (1992): The contents of fructose, citric cid, cid phosphtse, proteins nd electrolytes in the ccessory sex glnd secretions of the mle golden hmster. Int J Androl 16: Chow PH, Dockery P, Cheung MPL, S (1994): Effects of mle ccessory sex glnds on fertiliztion, polyspermy nd morphometry of erly embryos in the golden hmster. Int J Androl 17:

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