Seminal plasma proteins of fertile and infertile men analyzed by two dimensional electrophoresis

Samaan et al.

. December 1987

Am JObslet Gyneco l

,

I I. 12. 13.

14. 15.

feta l pig: effee ts of ehronie hyperinsulinemia , j Endoerino I 1983;96:107- 14. j ost A. Experi enees de deea pitat ion de l'e mb ryo n de lapin. Aead Sei Paris 1947;225:322-4. j ack,PMB, Milner RDG. Effect of d ecapi tatio n and ACT H on soma tie de velopment of the rabbit fetus. Biol Neonate 1975;26 : 195-204. Daughad ay WH, PhiIlips L S, Mueller MC. The effect of insulin and grow th hormon e o n the release of somatomed in by the isolated ra t liver. End oerinology 1976;98:1214 . WiIliam s RL, Creasy RK, Cu nningha m GC, Hawes WE, No rris RD, Tashiro M. Fetal growth an d pe rin atal viability in Californi a. Obstet Gynecol 1982;59:624-32 . Sam aan NA, Yen SCC, Friesen H , Pear son OH. Seru m

,

placen tal laetogen levels d uring pregn ancy in tro phoblastic disease. j Clin End ocrin ol Metab 1966;26: 1303. 16. Furl anetto RW, Underwood LE, Van Wyk jj, Hand erwerger '5.'Serum .immu noreactive soma tomedi n-C .is elevated late in pregn an cy. j Clin End oerin ol Metab 1980;47 :695. 17. Zap f j , Froesch ER. In sulin-like growth. faeto rs somatorned ins: str ucture, secr etion , biological aetions and ph ysiologieal role. Horm on e Res '1986;24: 121-30. 18. Sam aan NA, Vassilopoulo u-Sellin R, Schultz PN, Rivera ME, Held B. No nsu p pressible insulin-like activity and somatom edin C levels in norm al pregn ant wornen , in pregnant wornen with gestational diabetes, and in u rnbilieal cord blood of mature and prernature infan ts. AMj OBSTET GY NECOL 1985;153:457 -6 1.

Seminal plasma proteins of fertile and infertile men analyzed by two-dimensional electrophoresis Ramchand~a R. Ayyagari, M.D., Asgerally T. Fazleabas, Ph.n., ~nd M. Yusoff Dawood, M.D. Chicago, Illinois Sperrn-tres semmal plasma frorn seminal fluid.ejaculate of fertile arid infertile men obtained in the presenceand absence of aprotinin(500 kallikrein inhibitor units per milliliter} was analyzed by two-dlmenslonal electrophoresls 10Uowed by silver statninq, To evaluats postliquefaction proteo lytic breakdown of seminal plasma protelns, protease lnhibitors were addedto the semen at 15, 30,and 60 rnlnutes after ejaeulation. Most seminaI plasma proteins tn normospermie men(n := 4} had molecu lar weights 01 30;000 to 70,000 and were slrnüar to those present in serum. The rnajor non-serUm protein in semmal plasrna 01 all men was a basic produet with an approxirnate molee~lar weight of 40,000. A group of protems (molecular weights = 20,000 to 23,000) in seminal plasma analyzed immediately after Iiquelaetion was detected in oligospermie men (n = 4) but not in norrnosperrnic men (n = 4) or azoospermie men (n = 4). When protease inhibitor was added to normospermic semen at ~15 minutes after Iiquefaction, these protelns (rnolecular weight, = 20,000 to 23,000) and another group of proteins (moleeular weights = 40,000 to 43,000) were readily identifiable but were further enhaneed in the absence 01 protease inhibitors. These findings suggest that oligosperm ie men may have accelerated proteolysis of sperrn or seminal plasma proteins that may contribute to subfertility. (AM J OaSTET G VNECOl 1987;157:1528-33 .)

Key words: Seminal pla sma proteins, oligos perm ia, azoos pe rm ia, two-dimensional elect ro p horesis, su bfertility Seme n ana lysis co ntinues to be a p rima ry laborator y method of evaluating male fe rti lity d espite its paar pred ictive value ex cept for th ose men with severe abnormalities. More re cently, th e ham ster zo na -fr ee oocy te From the Division of R eproductive Endocrinology, Department of Obstetrics anti Gynecology, University of Illinois College of Medicine. Supported in part by a grant from the BR SG Lola Wilson R esearch Fund. Pr esented at the Th irty-third Annual M eeting of the Society [or Gynecologic Investigation, Toronto, Ontario, Canada, M arch 19-22 , 1986. R eprint requests: M. Yusoff Dawood, M .D., Department ofObstetrics and Gynecology, University of Illinois College ofMedicine, 840 S. W ood St., Chicago, IL 60 61 2 .

1528

penetration test and in vitro eer vieal mu eus penetration test have add ed ne w dimensions to the evaluati on of male ga metes. However , these tests also are not co nsiste n tly reliable . H uman se minal pla sma has 18 to 55 gm protein per lite r " 2 with 40 % to 50 % derived fro m the se minal vesicles, 35 % to 40 % fro m the p rostat e, an d the re ma ind er fr om other accessory glands . A flu id such as huma n semina l plasm a with its co m plex composition and the prorein contribution from the ma le reproductive traet is likel y to h ave an effe ct on sperm survival and quality. A few lim itcd stu d ies have examine d the electrophoretic patte ras of the proteins in h um an seminal plasma," but in terpretation of the data

Seminal plasma proteins and male fertility

Volume 157 Num ber 6

is diffi eult sinee protease inhibitors were not ad de d to the semen exa rnined . Basie proteins in human seminal plasma have been shown to unelergo rapid proteolysis 15 minutes after eja culation."To d eterrnine if there are differenees in the protein profile of seminal pla sma of fertile and subfer tile men , we performeel a study with two-elimensional eleetrophoresis to map the aeidi e and basie seminal plasma proteins with anel with out the addition of the protease inhibitor aprotinin to the semen of normal , oligospe rmie, and azo ospermic men. This article re po rt s the findings of o ur study. Material and methods

Electrophoresis supplies, Bis-acr ylamide and N,N, N ' ,N ' -te trameth yle thylened iamine were obtained from Biorad Lab oratories, Richmond , Cali f., and acry lamid e was a product of Serva Biochemieals. He idelberg, Germany ; N,N '-diethyl-tartardiamide was purchased from Nati onal Diagnostics, Somerville, N.]., and amp holines were fr om LKB , Uppsala, Sweden. Coomassie blue R250 , sod iu m dod ecyl su lfa re, urea, d ith iothreitol, protein sta nd ards, 2ß·mercaptoethanol , agarose, and all other inorganic chemieals of rea gent grade or better were products of Sigma Chemieal Co., Sc Louis , Mo. The protease inhibitor aprotinin was also purehaseel from Sigma Chemica l Co. Subjects and patients. Male volu nteers 18 to 28 yea rs of age were reeruited from our Infertility Clinic . All subjeets gave eonsent to provide semen sampies. Normal men were defined as th ose who had fathered ch ildren and had two or more normal semen anal yses at intervals o f at least a month (spe r m co u nt > 20 rnillion /mI , > 50% motiIit y, and < 40% sperm with abnormal morphologie features). Oligospermie men were thos e who had a history o f infertility, three or more semen analyses ~ 1 month apa rt with a spe rm count of <20 million/mI , >50% motility, and > 50% sperm with normal morphologie features. Azoospermie men were those who had a history of infertility and no sperm in the ejaculate but who had fru ctose in the seminal plasma on at least two semen analyses ~ 1 month apart. Four normospermie, four oligospe r mie, and four azoo spermie men were stud ied. Collection of semen samples. The su bjeets in the study were asked to abstain from sexual activity for at least 36 hours be for e providing a semen sam pie . Ejaeulates were eollected by masturbation eit her into ster ile con tainers without protease inhibitors or into containers th at had previously been coated with 1750 kaIIikrein inhibitor units of ap rotinin . After liquefaction, a small aliquot of eae h of th e ejaeulates was set asid e for routine semen ana lysis. T he semen sampies were held at room temperature for 0, 15, 30, or 60 minutes after liquefacti on. An aliquot o f 250 to 500 ~l (depend ing on the volume of the ejaeulate) was rernoved fr orn eaeh ejae-

1529

ulate and the spe r ma tozoa were separated from the seminal plasma by centrifugation in a micr ocentrifuge (1O,OOOg x 10 minutes). Ejaculates obtained in the absence of any a protin in were further divided into two aliqu ots . One aliquot waskept at room temperature for 15, 30, o r 60 minutes while ap ro tinin (500 kallikrein inhibitor units/ml ) was added to the ot her at similar time intervals, then mixed in a vortex and cen tr ifugeel. The seminal plasma obtained at eaeh tim e point for eaeh treatment was separated from the sper m after een tri fug atio n anel stored at - 70° C. The protein co ncentration in the seminal plasrna obtaineel at eaeh time point was determined by the method of Lowry et al." Two-dimensional polyacrylamiele gel eIectrophoresis. Sam pies of seminal plasma (300 ~g protein) were dis solveel in 75 ~l of 5 mmol/L potassium carbonate eontaining 9.3 mo l/L urea, 2% (vol/vol) NP-40, and 0.5% (w/vol) dithiothreitol for analysis by twodimensional polyacrylamide gel eleetrophoresis. The sa mpies were analyied Ly isoeleetric focusing aecord ing to the method of O'Farrell' as modified by Horst et al." The proteins (p I < 8.0) were analyzed by isoelectric focusing in the first dimension in 4 %(w /vol) aerylamid e gels containing ampholines (3.5 to 10,5 to 7, and 9 to 11, 50 : 35: 15 by volu me, respectively). After isoelectric focusing was performed , the sam pIes were an alyzed in the second dimension by sod iu m d odec yl sulfate gel electrophoresis." The eq uilibrated electrofocused gels were overlaid on 10% (w/vol) acryl amieIe slab gels and eIeetrophoresis was performed toward the ano d e. The slabs were fixed in two ehanges o f ethanol, acetie aeid , and water (40 : 7 : 53 by volume, respeetively), stained with 0.12 5% Coomassie blue R-25 0, destained, and photographed. The gels of serninal plasma proteins were then stained with silver .!" Results

In the seminal specimens from all male subjects studied, the maj or non-serum protein present in seminal plasma was a basic protein with an approximate moleeular weight of 40 ,000. Most of the seminal plasma proteins present in normospermic men had molecular weights of 30,000 to 70,000 and were similar to th ose present in the serum ( Fig. I). In oligospe r mie men , a group of proteins with 010lecular weights of 20,000 to 23,00 0 was present in the semina l plasma immed iately afte r Iiquefaction. However, thes e proteins were not deteeted in sem inal pla sm a of either no rmospermie or azoospe r mie men at the sa me time point ( Fig. I). Fig. 2 shows the silver-stained two-dimensi on al electrophoretic gels of semina l plas ma fro m normal men and the e ffect of proteolysis at ro om temperature on the ap pearanc e of the group of proteins (moleeular weights 20,000 to 23,000). When the protease inhibitor

1530 Ayyagari, Fazleabas, and Dawood

December 1987 Am J Obstet Gynecol

pH

6 .1

•

3 ·5

•

MW x 10' 3

"' 6 8 "' 5 8

"'4 3

...30

...14

Fig. 1. Silver-stained two-di me nsio nal ele ctr o ph oret ic ge ls of seminal plasma fr o m normospermie (A) , oligospe r mie (B) , and azoos perm ie (C) men and of human plas ma (D ). Sern inal fluid ejaculates

were eolleeted in the presen ee of pro tease inhi bitor. A grou p o f prote ins with moleeular weights of 20,000 to 23,000

(J) is present in semi nal plasma fr om oligosper mie men (8) bu t is a bsent in

normospermie (A) and azoospermie me n (C). The a rrow indi cates th e basic prorein presenl in seminal plasrna but not in serum .

a protm m was not ad ded to the eja cu late after liquefac tion, th e group 0[. proteins (molecula r weights 20,000 to 23,000) th at was previousl y detected only in se rnina l plasrria of oligospermie men ( Fig. I, B ) appearedand iricreased in intensity o ver time . In ad d ition , another group of p rotein s (m olecu la r weigh ts 40 ,000 to 43,000) also appeared in normal seminal flui d with this treatme nt. When prot eol ysis of semen from normospermic me n was in h ibited -by the addition of th e p ro te ase inhibito r a protinin at 15, 30 , and 60 min utes after Iiquefaction, the two gro ups of proteins (molecular weights 20 ,000 to 23,000) that we re present in normal se m en afte r proteolysis were then also detectable by

two-d irnensional electro p ho resis ( Fig. 3). Ho we ve r, co m pa red with Fig . 2, the inte nsity .o f these p rot eins was significantly d im in ished o ve r time ( Fig. 3). Comment

Pr evious lim ited studies on sem ina l p lasma pro teins hav e examined only the semen of normal me n.>" Usin g two -dimensiona l gel electrophoresis, we foun d that th e major no n-sse r u m protein present in the semina l plasma o f both fe rti le and subfertile men was a basic prorein with a mol ecul a r weight of 40,000 . This is simi lar to the earlier repo rts o f sernen from normal me n .t" Of greate r significance, ho we ver, was o u r finding th at

Seminal plasma proteins and male fertility

Volume 157 Number 6

pH

___________ 6..1

.

3 ·5 .

1531

MWx 10- 3

Fig. 2. Silver-stained gels after two-dimensional electrophoresis of seminal plasma from norrnosperrnie men. Ejaculates were collected in the absence of the protease inhibitor aprotinin and kept at room temperature for 0 (A), 15 (B), 30 (e), and 60 (D) rninutes after liquefaction. The group of proteins with molendar weights of 20,000 to 23,000

(j) that was present in Fig. I, B, appeared and

increased in intensity over time after liquefaction. In addition, another group of proteins with malendar weights of 40,000 to 43,000 ( A_A) also appeared and increased in intensity over time.

a group of proteins with rnolecular weights of 20,000 to 23,000 is present in the seminal plasma of oligospermie men if analysis is done immediately after liquefaetion but not in the seminal plasma of normal or azoospermie men. Nevertheless, when a protease inhibitor such as aprotinin is added to normal semen 15 01' more minutes after liquefaction, these proteins (molecular weights = 20,000 to 23,000) are readily identified. In addition, another group of proteins with moleeular weights of 40,000 to 43,000 also beeame identifiabIe in normal seminal fluid treated with the protease inhibitor. Furthermore, the identifieation of these two groups of proteins (rnolecular weights =

20,000 to 23,000 and 40,000 to 43,(00) was enhaneecI in the absence of protease inhibitors. Thus, a group of proteins with rnolecular weights of 20,000 to 23,000 is readily deteeted in seminal plasma immediately after ejaeulation and liquefaetion in oligospermie individuals. This group of proteins (moleeular weights = 20,000 to 23,000) together with another group (rnoIeeular weights = 40,000 to 43,000) are deteetable in the semen of norrnosperrnic individuals only when proteolytie aetivity is not suppressed. These proteins could not be identified in the semen of azoospermie men. The proteins with moleeuIar weights of 20,000 to 23,000 are most likely the produets of aeeelerated pro-

1532 Ayyagari. Fazleabas, and Dawood

December J987 Am JObslet C yne col

MW x 10-3

49 2 <16 8 <1 58 <14 3

<1 30

<114

Fig. 3. Silver-stained two-d irne nsio nal electropho reric gels of semina! plasma fro m no rm osper mie rnen. The ejaculates were collec ted in the abse nce of the protease in hibito r a pro tinin th at was ad ded at 15, 30, and 60 minutes after liquefaet ion. The gr ollp of proteins with molecular weights of 20,000 to 23 ,000

(J)

that was found in oligospermie men in Fig. I , B, and u nd er the condition s in Fig . 2

in normospermie men as weil as the other gr oup o f proteins with rnole cu lar weights of 40,000 to 43 ,000 ( . -4 ) that appeared under the conditions in Fig. 2 is now present but dimin ished in intensity over time as eompared with Fig. 2.

teolysis and originate from spermatozoa that u nclergo prot eolytic degra datio n. The a bse nce of these proteins in the seminal p lasma of azoospermie ind ivid uals with or without varying degrees of p ro teol ytic in hibition suggests the spermatozoa as th e souree of these proteins. The ra pid appearance and eas y deteetion of these proteins in semina l plasma of oligospermie men eombined wit h enhaneement of their deteetion in normal seminal plasma whe n prot eolysis is not in hibited strongly su p por t the role of acceleratcdpro tcolysis for the generatio n of these proteins. T herefore, the serninal flu id of oligospermie men has a p paren tly accel -

erated proteolytie activit y. What is less clear, however, is whether such a p pa rently aeeelerated proteolytie activity is d ue to ine reased levels of p roteolytie enzyme(s) activity and/or increased suseeptibility of spermatozoa to proteolytic d egradation in th e sernen of oligospermie men . Our hypothesis and ex plana tion ean an d should be further explored in inv estigations on previously fertile rne n who have undergone vasectorny, but this was not within the original seope of th is study. While p revious studies hav e examined seminal p lasma from normal men and as many as 200 protein bands were found on th e two-di rne nsional eleetropho-

Volume 157 Number 6

resis,"" similar analyses have not been reported for oligospermie and azoospermie rnen . Nonetheless, interpretation of the data from the se stud ies>' was eomplieated by the fact that protease inhibitors were not added to the semen eolleeted. The basic proteins in seminal plasma undergo rapid proteolysis 15 or more minutes after ejaculation ." Our findings show that proteins of 20,000 to 23,000, which are products of aeceIerated proteolysis in oligospermie rnen, also appear in normospermie serninal plasma, at a slower but normal rate of proteolysis. Furthermore, another group of proteins with moleeular weights of 40,000 to 43,0 00 is also a produet of the proteolysis of normal semen but does not show up readily in oligospermie seminal plasma . Therefore, there is a need to examine proteolysis and its inhibition and the possible contribution by sper m degradation to the appearanee of some proteins in serninal plasma by studying seminal plasma of individuals with normospermia, oligospermia, and azoospermia as weil as the effeet of protease inhibitors on the presenee and detection of any proteins. It is not c1ear whether the proteins (moleeular weights = 20,000 to 23,000) that we found in the seminal plasma of oligospermie men reflect subfertility or give rise to infertility. If proteolysis of spermatozoa is accelerated in the ejaeulates of oligospermie rnen , the number of spermatozoa available for ascent into the female reproductive tr act eould be redueed . On the other hand, such accelerated proteolysis ma y eliminate abnormal or nonviable spermatozoa as a means of ensuring selection of the more optimal spermatozoa, sinee proteolysis also oceurs in normal semen but at a slower rate. Further studies are ne cessary to clarify these rnechanisms.

Seminal plasma proteins and male fertility

1533

Ineonclusion, we have found aeeelerated proteolysis in the semen of oligospermie men and it is readily demonstrable earl y after ejaeulation and Iiquefaetion by two-dimensional gel eleetrophoresis. This derangement eould be aeontributory factor to the infertility of oligospermie men . REFERENCES 1. Qu inlivan WLG. Analys is of the proteins in human seminal plasma. Arch Biochem Biophys 1968;127 :680-7 . 2. Tauber PF, Zaneveld LJD, Propping D, Schumacher GFB. Cornponents of human split eja culates. 1. Sper rnatozoa, fructose , immunoglobulins, albumin, Iactoferrin, transferrin and othe r plasma proteins, J Reprod Fertil 1975; 43 :249-67. 3. Lizana j, Eneroth P, Bygdeman M. Anal ysis of human seminal plasm a proteins by isoelectri c focusing , twod imensional electrophoresis and related immunoche mical techniques [Abstract 143]. Clin e hern 1981 ;27:1052. 4. Yuan j H, Wortham]WE. Characterization of hurnan serninal plasma protein using high resolution two-dirnensional elect rophoresis [Abstract 144]. CIin Chem 1981 ;27 :1053. _ 5. Edwards JJ, Toll aksen SL, Anderson NG. Proteins of human sernen. 1. two-d imensional mapping of human seminal fluid . Clin Chem 1981;27 :1335-40. 6. Lowry OH , Rosebrough NJ, Farr AL.:Rand all RJ. Protein mea surements with the Folin ph enol reagent. J Bio! Chern 1951; 193:265-7 5. 7. O'Farrell PH . High-resolution two-dimensional electro phoresis of prorein . J Biol Ch em 1975;250:4007-21 . 8. Horst MN, Basha SMM, Baumbach GA, Mansfield EH , Roberts RM. Alkaline urea solubilization, twodimensional electrophoresis and lectin stain ing of mammal ian cell plasm a membrane and plant seed pr oteins. Anal Bioehern 1980 ;102 :399- 408. 9. Laemmli UK. Cleavag e o f structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227 :680-5 . 10. Wra y W, Boulihas T , Wra y VP, Han cock R. Silver stainin g of proteins in polyacrylamid e gels. Anal Bioche m 1981 ; 118:197-203.