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Studies on proteins of seminal fluid from the vasa deferentia of the cock, Gallus gallus L.
ht. 3. Biochem. STUDIES ON PROTEINS OF SEMINAL FLUID FROM THE VASA DEFERENTIA OF THE COCK, GALLUS GALLUS L. A. STRATIL
(-
IONw., rg6g)
ABSTRACT ~.Bymeansofimmuno-Cl~theptaenn ofatleast x2com ncmtsw2s detectedin~pIarrnoofdomaticmorta:(CollusgallwL),4_5ofw~ wCrC$ulti*cEY .galialuydifkcntfrombloodbloodprotCins. 2. Gel flhratk on Se@adex Gnoo is a suitable method for the fiactio~lrtionof seminal nlaanrpmteina 3. The comparison of e&cts of nb on iron-binding proteins of seminal ~#erul&andeeg-whitCshowatbatsa5inalplasmaplPlrmaerrilisarCrCptwCntCd bymolecuhswith2, x,2ndorhduaof&ilicacidperprotCinmokcule.
ALTEIouo5i the cbanical compositicmof cock seminal plasma has beal inkIlsivCly studied (for rtfw sa Lake, I g66), comparatively little data have been publishedabout ia protein composition It is known literature that the ‘transp~t hid’
hm
contains afhr clectrophoresis the same fractions as the serum @vii‘957). In cock 8Ukli4phglPathCpnsenCCof‘traarfarins’ and ‘al-’ has been detected; both these proteins, howwer, difk in certain rcspcca km the comsponding proteins in the serum (Ogden, ~$2; Strati& xg68a, b, c). We have carried out a number of investiganions on the protein composition of cock 8cminalplasmaatldthercsultsofthcsC StUdiCS8l-CpraentcdiIlthiSpaper. MATZRIALS AND METHODS ThC8$m8lslnCd(caulls~L),tbcnunner ofcoikcwmofaxklCmin81fiuid(aemin81p18uMy havCbCal ~?~!&C*~&~=& T @cl. IlnmmzAwx The production ofantibodiCa wa8 hducai by immuni2ation of rabbits with SP, twkular fluid, and saum of cocks, rapectively. Imm~tions
were pcrhmcd only with frahly gained fluids without preceding storage. Each rabbit was given iI+tiom of2-3 ml. antigen aubcutallwsiy atweeklyintervk AweekaftCrthehtinjection thCbloodwzucohxed. Rc-immuhations with twoi+ctionsofant@lwaea&Cdout&.Cr2n intcrvplof3monthafiwntheprC&hgimmunimthL ThCbC8tquality2ntkrafkoolthefimt 2nd aeamd re-immunhations were chooen for immun~~ Micr&ilmo-d~& (SchCidCggCr, Ig55)wascaniCdoutinasimihrwaytothatdes&bCdbyB rummentedt-Hatnen (xg67). Abrorp tiolp~pCIkmCdCithCrill8tUt-&C(otlCpZUtd bsenmlplusonepartofantigCnwCrCincub8tCd 2 a temperature of 37°C. for I hour! the wu applied to the ~tibody groove and the prep8ratioaw88iCftfcrr I how at 370 c.; the rCmn8ntofthe2z&genw8sthulrcmov& thCalltibodygmovC%ushalwith~gpacalt NaCl, and 6Ued with antiserum). AUTOWlOt3~ IO & “FCC& solution ( - activity 10 pc.) wCrCaddCdto4o~sP. TiP nmpla were kept 15 miduti at 37” C bdixe 8pplying them to thcantigalwCus. AftCrdxyingtheprCpar8tiona were attachal to an X-ray Elm and &peud for 14 hyk
SEMINAL. FLUID
197% 1s 728-734 Emuus
SThwm0
The detection of esterax on immuno-electrophomtic preparations was carried out affording to the method of Kamby+lis, Johnson, ~~l&hardson (I g68), as mod&d by Kubek I . STARCH-GEL
&ECTltOPHOXESlS
electrophoresis (horizontal arrangement) was essentially the same as described by Stratil (1967). Starch+
~RACTIONM7ON
OF
SP PROTEINS
The pool of SP (7.5 ml.) which had been obtained after centrifugati~n of spermatozoa
FIG. I .-Schematic
PROTEINS
729
TREATMENT OF SP AMINmAsE
TRMWERRXNS WITH NE~JR-
Five mg. lyophilized Fraction C (following chromatography of seminal plasma with Tf&) were dissolved in I ml. 0.2 A4 acetate buffer, pH 5.5, with ~04 M CaCl,. Added were ~4 ml. neuraminidase from Vibrio choimac (Koch-Light Laboratories Ltd.; 5oo unia per ml.) and the mixture was incubated at 37” C. for 48 hours. After incubation the sample was desalted on a Sephadex G-25 column with o5 per cent NH,OH as eluant and lyophilized. The lyophilizate was dissolved in 50 gl. Tris-citrate bu&r used for preparing starch-gel. The sample was then exposed to starch-gel elecuophoresis.
illustration of immuno-electrophoretic pattern of cock seminal plasma (SP). body: anti-SP serum.
Anti-
FIG. 2.-Immuno-electrophoresis of cock SP by use of homologous antiserum. Upper well: Concentrated SP (8 mg. lyophiliaate of high molecular weight portion after gel filtration on Sephadex G-25, solved in loo pl. buffet). Lower well: Normal SP. (5000 r.p.m. for 30 minute3 at 10’ C.) from the contena of vasa deferentia of freshly killed cocks was desalted on a Sephadex column G-25 using ~5 per cent NH,OH as eluant. The high molecular weight portion was Iyophiliaed and dissolved in 7.5 ml. Tris-HCl eluant buf%r and applied to the column (5 sq. cm. x g7 cm.) with Sephadex G-loo. A buffer consisting of 0. I M Tris-HCl, /JH 8.0, I so M NaCl, and 0’02 per cent sodium aaide was used for elution. The flow-rate was x7.7 ml. per hour; fractions were collected at lo-minute intervals. The protein contents was determined spectrophotometrically at 280 mp.
The control sample was treated in an analogous manner without incubation with neumminidase. RESULTS IMMUNO-ELECTROPHORE~I~ OF SP AND TESTICULAR FLUID OF COCKS By the use of homologous antiserum we have proved up to I 2 arcs in SP (Fig. I ). The contents of some proteins in unconcentrated SP is very low so that the number of arcs observed is smaller than that of more
730
STRATIL
concentrated SP. Fig. z shows the difference between concentrated and normal SP. A total of 42 samples have been analysed. Always present were arcs A (albumin), Tf (transferrin), and 8 A which are very strong and characteristic; other weak fractions were not observed on some slides because of their very low intensity. In some cases we have observed differences in number and intensity of arcs, especially in the ax-us and pr-regions. SP tran&hrrins showed the capacity of bindiug “Fe, both when antiserum against
fat.
J.
Biochem.
When we used antiserum against SP absorbed by cock serum for the precipitation of SP proteins, an arc in the a1 region appeared on the preparation, further I -2 arcs in the & region and arcs 6* and 6 ,. A similar pattern was achieved when testicular fluid was used for absorption; testicular f&rid, however, also absorbs the antibody against one protein component migrating in the fir region. There are probably some more components ; the antibodies against them do not seem to be absorbed either by serum or
FIG. 3,--Autoradiographic iclcnti6cation of’ SP trzdhhs Mekti with ‘*Fe. Antibody: anti-SP serum. A, Amid0 black s&ring. The tmn&rin are is dcdgnated by an armw. 6, Autoradiography of the same preparation. SP (Fig. 3) and antiserum against cock serum were used, Arc y,, was observed only on seved slides ; it was usually very weak, doubled, and diffie. It was more intensive only on slides with concentrated SP. The fmtion 6 * reaches far into the cathodic field and is localized more cathodically than the y-globulin arc of the serum. Estemse staining resulted in the formation of one very intensive arc in the albumin region, the cathodic part of which often trails into the &-region (Fig. 4). This arc may also be detected by the use of antiserum against cock serum.
by testicular fluid. The intensity of the corresponding arcs may be so weak that they cannot be observed on the preparations. Im.muno-electrophoresis of testicular &rid by the use of homologous antiserum led to the detection of components uxresponding to serum antigens; their intensity was, however, lower than in the reaction cock serum/anti-cock serum, and the preparations were not so clear. No arcs were observed when antiserum against tcstidar fluid was absorbed by cock serum and used against SP.
SKMINAL FLUID PROTEINS
‘970, 1
C. Immune-electrophoresis of Fraction A yielded no expressive ares {only traces of albumin, ~~-glob~n, and yglobulin). In Fraction B there is a strong y-globulin arc with another parallel weak fraction ; traces of albumin and some very weak arcs in
FRACTIONATION OF SP PROTEINS In order to get more detailed characterizadon of SP proteins and to obtain the transferrin fraction which was less contaminated by other proteins, we have used the method of gel filtration on Sephadex G-200. The elution pattern is shown in Fig. 5. The contents
of SP by use of homologous antiserum. Esterasc staihing.
FIG. q.-I~~o-elcctroph~
Go
260 ’
FIG. 5.-Gel
A
I ’
731
B
, I
3io
360 ,. C
’
40
D
’
E
filtration of cock SP proteins on Scphadex G-200. dcxwan is also shown (a).
of test-tubes were divided into 6 f&tions A, B, C, D, E, and F, and each fraction was desalted on Sephadex G-a5 in o-5 per cent NH,OH, lyophilized, and analysed immune-electrophoretieally. Peak A was eluted with void volume. This fraction is milk white, which is why it apparently causes high absorbancy. The amount of lyophilizate was about half of that in peak
the
al-a,
i&J
450
1.
I ’
ml.
4
F
Column testing by means of blue
regions
were
also
present
(Fig_ 6A), In the mentioned fraction esterase activity has been found reaching into Fraction C. After iyophilization, Fraction C is a pink substance which, after dissolving in Triscitrate buffer, forms a rust-brown fluid. This fraction contains albumin and transferrin in high concentration. Besides this pre-albumin
73’
STRATIL
also present and some very weak components in the a- and /S-regions (Fig. 66). During elcctrophoresis in agar gel, transferrin migrates as a rust-brown spot, and in starch-
is
ht.
J.
Btieh.
some other components in the u- and @ regions (Kg. 6C). The 6* component is characteristic for Fraction E ; then there are traces of
FIG. 6.-I~nmuno-electrophorcais of individual fractions after gel filtration of SP ptwchs. Antibody: anti-SP serum. The lower well on all slides contains concentrated SP, the upper wells contain the individual fiaction~~ A, Fraction B; 8, Fraction C; C, Fraction D; D, Fraction E. gd eiectrophoresis it forms at kast two rustbrown fktions in unstained gel. Fraction D contains albumin, weak arcs of prealbumin, transferrin, S*-component, and
albumin and eventuaiiy some other proteins in the a- and B_regions (Fig. 6D). A similar pattern may be found in Fraction F.
SEMINAL
197% 1
FLUID
733
PROTEINS
zone in untreated transferrins. Fractions I and 2 have the same kxahaation as the fractions of serum tran&rins and the localization of zone o corresponds to that of the main egg-white conalbumin fraction. The incubation of SP dialysed against 0.2 M acetate buf&r with 0.04 M Cat&
The effkct of ncuraminidase on SP transferrins is demonstrated in Figs. 7 and 8. Untreated transferrins are formed by three fractions, the strongest of which, zone o, migrates slowest, and the weakest, zone 2,
D-I
0
A
B
C
D
FXG. 8.-Schunatic diagram of iron-binding proteins of tbe &i&en serum, cock SP, and hen’s egg-white after starch-gel &ctrophorc&s (phenotype Tf*). A, W tran&krrins. B, SP transferrins not treated with n etlmmkidase. c, SP trans5zrrins treated with nemamkidase. D, Conalbumins of egg-white. In this sample only those ftactions are entered which correspond to conalbumins after saturation with Fe’+. The numbers of zone5 0, x and 2 designate the amotmt of sialic acid residues per protein molccuk
without the addition of neuraminidase for 48 hours at 38” C. had no effect on the starch-gel ekxtrophoretic pattern of tranr&rrins, DISCUSSION
A
0
c
D
E
F
Fro. 7.-Starch-g& electrophoresis showing the effect ofne uraminidase on SP transferrin fractions. C, Fraction C from the gel &ration, treated with ncuraminidast. D, Fraction C untreated. B; E, NormaJ SP, for comp&son’s sake the separation
of the serum (A) and the egg-white (F) are demonstrated. Ail sample3 were taken from animals of TfA/TfA genotype. The main fixtions of iron-binding proteins iu A, C, D, and F arc marked with a cross. migrates quickest to the anode during starchgel electrophoresis. Treated transferrins have only fractions o and I. Zone 2 is practically missing. The intensity of fraction I is substantially lower than that of the same
By means of immuno-electrophoresis a considerable number of protein components (at least I 2) was proved in SP of cock, and the presence of other minor components cannot be excluded. A number of proteins are antigenitally related to those of the serum (see also Stratii, rg68c) but some are specific for SP (at least 4-5), arc 6, being the most characteristic Arc y* probably represents serum yglobulin. Its exact identification was not possible because of its low intensity and diffuseness. The presence of this component in SP may be due to contamination with blood in some cases in spite of very carefX handling during SP collection. Gel filtration on Sephadcx G-200 is an advantageous method for the fractionation of SP proteins. It is evident that the first chromatography does not yield perfect purity
mum
734
oiindividual proteins, but some fractions can be used for further investigations, because their heterogeneity is much lower in comparison with unfktionated SP. It may be seen from the elution pattern that SP contains proteins of the most various molecular weights. The finding that component 6, has relatively low molecular weight (lower, e.g., than albumin) is of great interest. The most complete results were obtained for SP transferrins. This protein has the same genetic det ermination as serum transfenins and egg-white conalbumins, while it is phenotypically somewhat di&rent from both these proteins (Ogden, xg6z ; Strati& I g68a, b,c). Itwasalsofoundthatinmanyrespects SP transferrim resemble serum tran&enins and egg-white conalbumins by their properties (binding of ‘*Fe, pink colour of isolated tra&errins, rust-brown colouring of fractions in agar- and starch-gel electrophoresis). When treating serum transferrins with neuraminidase Williams (1962, q68) proved that transfdns have molecules with 2 or I: residues of sialic acid, respectively. Conalbumins do not contain sialic acid. If this finding is applied to explain the heterogeneity of SP tranaferrins, it is probable that they are represented by proteins without sialic acid (fraction o), with I residue (fraction I), and with 2 residues (fraction 2) of sialic acid per protein molecule, respectively. This conclusion is strongly supported by the results obtained a&r treatment of SP transferrins with neuraminidase. Acxum The
-
author thanks Mrs. M. Sulcovzk for valu-
able technical collaboration.
REFERENCES
M. P., JOHNSON, F. M., and R. H. (I g68), ‘ hozyme variability in soecia of the genus Dmsotihila. IV. Distributioxi of the ester&d in the’body tissues of D. akbichi and D. mtdiuri adults ‘, Biachm. Gent., x,
KMB~~ELLIS, bXARDBoN,
249-265.
A. (1g6g), ‘ hhiium polymorfkt+ch proteinov a enamqov kcvnkho s&a a niekto+h pohlavrqkh tekutinc&pat@ ‘, Thesis, CzechoSlovak Academy of Sciences, Laboratory of Physiology and Genetics of Animals, Libtchov. LAXE, P. E. (x966), ‘ Phyaiohgy and biochemistry in AakUnus in Rcpmductive ofpoultry =mQl’, E~YC~~~N, A.), vol. I,pp. 93-123. Kfrst~,
N-4
H. ( 1957; ‘ On the characteristics of the transparent fluid. II. An electrophoretic study of proteins of the trampamnt fluid ‘, 3. Fat. Agric. Kjushu Univ., II, 63-68. OGDEN, A. L. (q62), ‘ Ezprasion of the transferrin gene in tbe serum, uur-white and seminal fluid +eins of the a ‘, Mimmgr~ R@lwtonth#EigkkE8ifqmn~onAnimal Blood Gonb Rwmh. Liubliana. Yuaosiavia.
J. J. (I&&, ‘*Un; mi&m&hode de I’immun~&ctrophor&se ‘, ht. A&s A&Y appl. Immun., 7, x03-110. STUIU., A. (1g67), ‘ The effect of iron addition to avian egg-white on the behaviour of conalbumin &actions in star&gel electrophomsis’, &n@. Biochm. PM, no, 227-233. SBE&
--(q6&),‘Transfinrinandalbuxninlociin chiw Gak * L.‘, *, 36 I 13-121. - - (rg68b) ‘ carectclri, v+@q!$?~~ -mriddita protcixlll alqnc , slovak Academy of Scienca, Laboratory of Physiology and ik~etics of Animals, Libkhov. --f1o6&~.‘Proteinsoftheseminalfluidfrom the ;P; d&ens of cocks; their polymorphkm and relation to serum proteins’, Presented at XIth Conference on Blood Groups and Protein PoIymorphism in Animals, Warsaw, 1-6 July, x966. Ww, J. (I@), ‘ A comparison of conalbumin and tnnsfkrrin in the domestic fowl ‘, B&&m. 3.9 89,355-364. -(xg66), ‘A comen of giycopeptides from the ovotramfti and serum transferrio of the hen 0 Ibid., x0&57-67.
(-
IONw., rg6g)
ABSTRACT ~.Bymeansofimmuno-Cl~theptaenn ofatleast x2com ncmtsw2s detectedin~pIarrnoofdomaticmorta:(CollusgallwL),4_5ofw~ wCrC$ulti*cEY .galialuydifkcntfrombloodbloodprotCins. 2. Gel flhratk on Se@adex Gnoo is a suitable method for the fiactio~lrtionof seminal nlaanrpmteina 3. The comparison of e&cts of nb on iron-binding proteins of seminal ~#erul&andeeg-whitCshowatbatsa5inalplasmaplPlrmaerrilisarCrCptwCntCd bymolecuhswith2, x,2ndorhduaof&ilicacidperprotCinmokcule.
ALTEIouo5i the cbanical compositicmof cock seminal plasma has beal inkIlsivCly studied (for rtfw sa Lake, I g66), comparatively little data have been publishedabout ia protein composition It is known literature that the ‘transp~t hid’
hm
contains afhr clectrophoresis the same fractions as the serum @vii‘957). In cock 8Ukli4phglPathCpnsenCCof‘traarfarins’ and ‘al-’ has been detected; both these proteins, howwer, difk in certain rcspcca km the comsponding proteins in the serum (Ogden, ~$2; Strati& xg68a, b, c). We have carried out a number of investiganions on the protein composition of cock 8cminalplasmaatldthercsultsofthcsC StUdiCS8l-CpraentcdiIlthiSpaper. MATZRIALS AND METHODS ThC8$m8lslnCd(caulls~L),tbcnunner ofcoikcwmofaxklCmin81fiuid(aemin81p18uMy havCbCal ~?~!&C*~&~=& T @cl. IlnmmzAwx The production ofantibodiCa wa8 hducai by immuni2ation of rabbits with SP, twkular fluid, and saum of cocks, rapectively. Imm~tions
were pcrhmcd only with frahly gained fluids without preceding storage. Each rabbit was given iI+tiom of2-3 ml. antigen aubcutallwsiy atweeklyintervk AweekaftCrthehtinjection thCbloodwzucohxed. Rc-immuhations with twoi+ctionsofant@lwaea&Cdout&.Cr2n intcrvplof3monthafiwntheprC&hgimmunimthL ThCbC8tquality2ntkrafkoolthefimt 2nd aeamd re-immunhations were chooen for immun~~ Micr&ilmo-d~& (SchCidCggCr, Ig55)wascaniCdoutinasimihrwaytothatdes&bCdbyB rummentedt-Hatnen (xg67). Abrorp tiolp~pCIkmCdCithCrill8tUt-&C(otlCpZUtd bsenmlplusonepartofantigCnwCrCincub8tCd 2 a temperature of 37°C. for I hour! the wu applied to the ~tibody groove and the prep8ratioaw88iCftfcrr I how at 370 c.; the rCmn8ntofthe2z&genw8sthulrcmov& thCalltibodygmovC%ushalwith~gpacalt NaCl, and 6Ued with antiserum). AUTOWlOt3~ IO & “FCC& solution ( - activity 10 pc.) wCrCaddCdto4o~sP. TiP nmpla were kept 15 miduti at 37” C bdixe 8pplying them to thcantigalwCus. AftCrdxyingtheprCpar8tiona were attachal to an X-ray Elm and &peud for 14 hyk
SEMINAL. FLUID
197% 1s 728-734 Emuus
SThwm0
The detection of esterax on immuno-electrophomtic preparations was carried out affording to the method of Kamby+lis, Johnson, ~~l&hardson (I g68), as mod&d by Kubek I . STARCH-GEL
&ECTltOPHOXESlS
electrophoresis (horizontal arrangement) was essentially the same as described by Stratil (1967). Starch+
~RACTIONM7ON
OF
SP PROTEINS
The pool of SP (7.5 ml.) which had been obtained after centrifugati~n of spermatozoa
FIG. I .-Schematic
PROTEINS
729
TREATMENT OF SP AMINmAsE
TRMWERRXNS WITH NE~JR-
Five mg. lyophilized Fraction C (following chromatography of seminal plasma with Tf&) were dissolved in I ml. 0.2 A4 acetate buffer, pH 5.5, with ~04 M CaCl,. Added were ~4 ml. neuraminidase from Vibrio choimac (Koch-Light Laboratories Ltd.; 5oo unia per ml.) and the mixture was incubated at 37” C. for 48 hours. After incubation the sample was desalted on a Sephadex G-25 column with o5 per cent NH,OH as eluant and lyophilized. The lyophilizate was dissolved in 50 gl. Tris-citrate bu&r used for preparing starch-gel. The sample was then exposed to starch-gel elecuophoresis.
illustration of immuno-electrophoretic pattern of cock seminal plasma (SP). body: anti-SP serum.
Anti-
FIG. 2.-Immuno-electrophoresis of cock SP by use of homologous antiserum. Upper well: Concentrated SP (8 mg. lyophiliaate of high molecular weight portion after gel filtration on Sephadex G-25, solved in loo pl. buffet). Lower well: Normal SP. (5000 r.p.m. for 30 minute3 at 10’ C.) from the contena of vasa deferentia of freshly killed cocks was desalted on a Sephadex column G-25 using ~5 per cent NH,OH as eluant. The high molecular weight portion was Iyophiliaed and dissolved in 7.5 ml. Tris-HCl eluant buf%r and applied to the column (5 sq. cm. x g7 cm.) with Sephadex G-loo. A buffer consisting of 0. I M Tris-HCl, /JH 8.0, I so M NaCl, and 0’02 per cent sodium aaide was used for elution. The flow-rate was x7.7 ml. per hour; fractions were collected at lo-minute intervals. The protein contents was determined spectrophotometrically at 280 mp.
The control sample was treated in an analogous manner without incubation with neumminidase. RESULTS IMMUNO-ELECTROPHORE~I~ OF SP AND TESTICULAR FLUID OF COCKS By the use of homologous antiserum we have proved up to I 2 arcs in SP (Fig. I ). The contents of some proteins in unconcentrated SP is very low so that the number of arcs observed is smaller than that of more
730
STRATIL
concentrated SP. Fig. z shows the difference between concentrated and normal SP. A total of 42 samples have been analysed. Always present were arcs A (albumin), Tf (transferrin), and 8 A which are very strong and characteristic; other weak fractions were not observed on some slides because of their very low intensity. In some cases we have observed differences in number and intensity of arcs, especially in the ax-us and pr-regions. SP tran&hrrins showed the capacity of bindiug “Fe, both when antiserum against
fat.
J.
Biochem.
When we used antiserum against SP absorbed by cock serum for the precipitation of SP proteins, an arc in the a1 region appeared on the preparation, further I -2 arcs in the & region and arcs 6* and 6 ,. A similar pattern was achieved when testicular fluid was used for absorption; testicular f&rid, however, also absorbs the antibody against one protein component migrating in the fir region. There are probably some more components ; the antibodies against them do not seem to be absorbed either by serum or
FIG. 3,--Autoradiographic iclcnti6cation of’ SP trzdhhs Mekti with ‘*Fe. Antibody: anti-SP serum. A, Amid0 black s&ring. The tmn&rin are is dcdgnated by an armw. 6, Autoradiography of the same preparation. SP (Fig. 3) and antiserum against cock serum were used, Arc y,, was observed only on seved slides ; it was usually very weak, doubled, and diffie. It was more intensive only on slides with concentrated SP. The fmtion 6 * reaches far into the cathodic field and is localized more cathodically than the y-globulin arc of the serum. Estemse staining resulted in the formation of one very intensive arc in the albumin region, the cathodic part of which often trails into the &-region (Fig. 4). This arc may also be detected by the use of antiserum against cock serum.
by testicular fluid. The intensity of the corresponding arcs may be so weak that they cannot be observed on the preparations. Im.muno-electrophoresis of testicular &rid by the use of homologous antiserum led to the detection of components uxresponding to serum antigens; their intensity was, however, lower than in the reaction cock serum/anti-cock serum, and the preparations were not so clear. No arcs were observed when antiserum against tcstidar fluid was absorbed by cock serum and used against SP.
SKMINAL FLUID PROTEINS
‘970, 1
C. Immune-electrophoresis of Fraction A yielded no expressive ares {only traces of albumin, ~~-glob~n, and yglobulin). In Fraction B there is a strong y-globulin arc with another parallel weak fraction ; traces of albumin and some very weak arcs in
FRACTIONATION OF SP PROTEINS In order to get more detailed characterizadon of SP proteins and to obtain the transferrin fraction which was less contaminated by other proteins, we have used the method of gel filtration on Sephadex G-200. The elution pattern is shown in Fig. 5. The contents
of SP by use of homologous antiserum. Esterasc staihing.
FIG. q.-I~~o-elcctroph~
Go
260 ’
FIG. 5.-Gel
A
I ’
731
B
, I
3io
360 ,. C
’
40
D
’
E
filtration of cock SP proteins on Scphadex G-200. dcxwan is also shown (a).
of test-tubes were divided into 6 f&tions A, B, C, D, E, and F, and each fraction was desalted on Sephadex G-a5 in o-5 per cent NH,OH, lyophilized, and analysed immune-electrophoretieally. Peak A was eluted with void volume. This fraction is milk white, which is why it apparently causes high absorbancy. The amount of lyophilizate was about half of that in peak
the
al-a,
i&J
450
1.
I ’
ml.
4
F
Column testing by means of blue
regions
were
also
present
(Fig_ 6A), In the mentioned fraction esterase activity has been found reaching into Fraction C. After iyophilization, Fraction C is a pink substance which, after dissolving in Triscitrate buffer, forms a rust-brown fluid. This fraction contains albumin and transferrin in high concentration. Besides this pre-albumin
73’
STRATIL
also present and some very weak components in the a- and /S-regions (Fig. 66). During elcctrophoresis in agar gel, transferrin migrates as a rust-brown spot, and in starch-
is
ht.
J.
Btieh.
some other components in the u- and @ regions (Kg. 6C). The 6* component is characteristic for Fraction E ; then there are traces of
FIG. 6.-I~nmuno-electrophorcais of individual fractions after gel filtration of SP ptwchs. Antibody: anti-SP serum. The lower well on all slides contains concentrated SP, the upper wells contain the individual fiaction~~ A, Fraction B; 8, Fraction C; C, Fraction D; D, Fraction E. gd eiectrophoresis it forms at kast two rustbrown fktions in unstained gel. Fraction D contains albumin, weak arcs of prealbumin, transferrin, S*-component, and
albumin and eventuaiiy some other proteins in the a- and B_regions (Fig. 6D). A similar pattern may be found in Fraction F.
SEMINAL
197% 1
FLUID
733
PROTEINS
zone in untreated transferrins. Fractions I and 2 have the same kxahaation as the fractions of serum tran&rins and the localization of zone o corresponds to that of the main egg-white conalbumin fraction. The incubation of SP dialysed against 0.2 M acetate buf&r with 0.04 M Cat&
The effkct of ncuraminidase on SP transferrins is demonstrated in Figs. 7 and 8. Untreated transferrins are formed by three fractions, the strongest of which, zone o, migrates slowest, and the weakest, zone 2,
D-I
0
A
B
C
D
FXG. 8.-Schunatic diagram of iron-binding proteins of tbe &i&en serum, cock SP, and hen’s egg-white after starch-gel &ctrophorc&s (phenotype Tf*). A, W tran&krrins. B, SP transferrins not treated with n etlmmkidase. c, SP trans5zrrins treated with nemamkidase. D, Conalbumins of egg-white. In this sample only those ftactions are entered which correspond to conalbumins after saturation with Fe’+. The numbers of zone5 0, x and 2 designate the amotmt of sialic acid residues per protein molccuk
without the addition of neuraminidase for 48 hours at 38” C. had no effect on the starch-gel ekxtrophoretic pattern of tranr&rrins, DISCUSSION
A
0
c
D
E
F
Fro. 7.-Starch-g& electrophoresis showing the effect ofne uraminidase on SP transferrin fractions. C, Fraction C from the gel &ration, treated with ncuraminidast. D, Fraction C untreated. B; E, NormaJ SP, for comp&son’s sake the separation
of the serum (A) and the egg-white (F) are demonstrated. Ail sample3 were taken from animals of TfA/TfA genotype. The main fixtions of iron-binding proteins iu A, C, D, and F arc marked with a cross. migrates quickest to the anode during starchgel electrophoresis. Treated transferrins have only fractions o and I. Zone 2 is practically missing. The intensity of fraction I is substantially lower than that of the same
By means of immuno-electrophoresis a considerable number of protein components (at least I 2) was proved in SP of cock, and the presence of other minor components cannot be excluded. A number of proteins are antigenitally related to those of the serum (see also Stratii, rg68c) but some are specific for SP (at least 4-5), arc 6, being the most characteristic Arc y* probably represents serum yglobulin. Its exact identification was not possible because of its low intensity and diffuseness. The presence of this component in SP may be due to contamination with blood in some cases in spite of very carefX handling during SP collection. Gel filtration on Sephadcx G-200 is an advantageous method for the fractionation of SP proteins. It is evident that the first chromatography does not yield perfect purity
mum
734
oiindividual proteins, but some fractions can be used for further investigations, because their heterogeneity is much lower in comparison with unfktionated SP. It may be seen from the elution pattern that SP contains proteins of the most various molecular weights. The finding that component 6, has relatively low molecular weight (lower, e.g., than albumin) is of great interest. The most complete results were obtained for SP transferrins. This protein has the same genetic det ermination as serum transfenins and egg-white conalbumins, while it is phenotypically somewhat di&rent from both these proteins (Ogden, xg6z ; Strati& I g68a, b,c). Itwasalsofoundthatinmanyrespects SP transferrim resemble serum tran&enins and egg-white conalbumins by their properties (binding of ‘*Fe, pink colour of isolated tra&errins, rust-brown colouring of fractions in agar- and starch-gel electrophoresis). When treating serum transferrins with neuraminidase Williams (1962, q68) proved that transfdns have molecules with 2 or I: residues of sialic acid, respectively. Conalbumins do not contain sialic acid. If this finding is applied to explain the heterogeneity of SP tranaferrins, it is probable that they are represented by proteins without sialic acid (fraction o), with I residue (fraction I), and with 2 residues (fraction 2) of sialic acid per protein molecule, respectively. This conclusion is strongly supported by the results obtained a&r treatment of SP transferrins with neuraminidase. Acxum The
-
author thanks Mrs. M. Sulcovzk for valu-
able technical collaboration.
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M. P., JOHNSON, F. M., and R. H. (I g68), ‘ hozyme variability in soecia of the genus Dmsotihila. IV. Distributioxi of the ester&d in the’body tissues of D. akbichi and D. mtdiuri adults ‘, Biachm. Gent., x,
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A. (1g6g), ‘ hhiium polymorfkt+ch proteinov a enamqov kcvnkho s&a a niekto+h pohlavrqkh tekutinc&pat@ ‘, Thesis, CzechoSlovak Academy of Sciences, Laboratory of Physiology and Genetics of Animals, Libtchov. LAXE, P. E. (x966), ‘ Phyaiohgy and biochemistry in AakUnus in Rcpmductive ofpoultry =mQl’, E~YC~~~N, A.), vol. I,pp. 93-123. Kfrst~,
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H. ( 1957; ‘ On the characteristics of the transparent fluid. II. An electrophoretic study of proteins of the trampamnt fluid ‘, 3. Fat. Agric. Kjushu Univ., II, 63-68. OGDEN, A. L. (q62), ‘ Ezprasion of the transferrin gene in tbe serum, uur-white and seminal fluid +eins of the a ‘, Mimmgr~ R@lwtonth#EigkkE8ifqmn~onAnimal Blood Gonb Rwmh. Liubliana. Yuaosiavia.
J. J. (I&&, ‘*Un; mi&m&hode de I’immun~&ctrophor&se ‘, ht. A&s A&Y appl. Immun., 7, x03-110. STUIU., A. (1g67), ‘ The effect of iron addition to avian egg-white on the behaviour of conalbumin &actions in star&gel electrophomsis’, &n@. Biochm. PM, no, 227-233. SBE&
--(q6&),‘Transfinrinandalbuxninlociin chiw Gak * L.‘, *, 36 I 13-121. - - (rg68b) ‘ carectclri, v+@q!$?~~ -mriddita protcixlll alqnc , slovak Academy of Scienca, Laboratory of Physiology and ik~etics of Animals, Libkhov. --f1o6&~.‘Proteinsoftheseminalfluidfrom the ;P; d&ens of cocks; their polymorphkm and relation to serum proteins’, Presented at XIth Conference on Blood Groups and Protein PoIymorphism in Animals, Warsaw, 1-6 July, x966. Ww, J. (I@), ‘ A comparison of conalbumin and tnnsfkrrin in the domestic fowl ‘, B&&m. 3.9 89,355-364. -(xg66), ‘A comen of giycopeptides from the ovotramfti and serum transferrio of the hen 0 Ibid., x0&57-67.