Indi an Journ al of Experimen tal Biology Vol. 40, March 2002, pp. 288-295

Immunological identification of two female-specific proteins from the plasma of Indian freshwater murrel, Channa punctatus (Bloch)

Neeta Sehga l & S V Goswami *

Department of Zoology, Un iversity of Delhi , De lhi 11 0007, India

Received 23 Nuvembe r 2000; revised 25 Septelllber 2001

Ex istence of a non-phosphorylated fem ale-specific protein (FS II ), in add iti on to phosphoryla ted vi tellogeni n (F I), in the plasma of murrel by exogcnous administ ration of estradiol- 17fS is reported. Polyspecific rabbi t ant ibod ies werc raised against estrogen-inducible mu rrel plasma proteins. Thi s ant iserum was absorbed with normal ma le serum in ordcr to obtain fcmale- spec ific anti scrum (FSAS ). Radi al immunodiffusion stud ies suggested that both the proteins (FS I and FS II ) were present in the plasma of Er treatcd and normal vitell ogenic females and in the ovarian homogenate from grav id females, but absent in norma l male plasma . Au toradi ographic experi ments demonstrated that phosphorus moiety was attached wit h FS I only. Further, immunoe lec trophoreti c ana lysis and peptide maps supported the observation that FS I and FS II werc di screte, unrelated female-specific proteins.

Vitell ogeni n, the egg yolk precursor, has been isolated from the blood of several fish species l

-6

. It is an estro­gen-inducible fema le-spec i fic multi component pro­tein ; present in the adult vi tellogenic females but ab­sent in males and in immature non-vitellogenic fe­males7

-9

. In addition, cDNA sequence fo r vitellogenin has been cloned and analysed9

. A characteri stic fea­ture of vitellogenin molecul e is the presence of alkal i­labi le phosphorus that permits its in vivo labelling with radiolabelled phosphorus. This characteristic has been employed by most investigators for identi fica­tion and estimation of vitell ogenin in the blood of several fishes and have led to the belief that vitel­logenin is the sole female-spec ific protein in the fi sh blood. Estrogen induces, not only the synthesis of vi­tell ogenin, but also of lipoproteins, egg envelope proteins that lack phosphoru s1o-14

. Obviously , these sex-spec ific, estrogen inducible non-phosphoryl ated proteins cannot be detected by these conventional techniques .

Immunology, whi ch offers analytical tools of ex­qui site prec ision, has been applied to develop immu­noassays (RIA and ELISA) for measurement of plasma vite llogenin, to establish precursor-product relationship between plasma vitellogenin and egg­yolk proteins, and to localize vitellogenin in fi sh

Correspondent author: Phone: 0 11-7667443 Fax: 0 11-7667524 e-mail: svgoswami@hotmai l.com

hepatocytes. Immunologica l techniques can also be employed fo r detecti on of female-specific pl asma pro­te ins that, unli ke vitell ogenin, lack phosphorus.

Present communicati on reports on immunolog ical identi fication of a fe male-specific protei n, in additi on to vitellogenin , in the blood of the Indian freshwater murrel, Channa punctatus, by using speci fi c antisera.

Materials and Methods Collection and care of flsh -Adult specimens of

C. punctatus (body weight 100-1 50 g) were collected from the backwaters of ri ver Yamuna around Delhi (Lat. 28°35' N, Long. 77° 12' E) and maintained at 25°± 1 ° C and a photoperiod of 12L: 120 fo r at least 7 days prior to use in experiments l5

. Fishes were fed ad libitum every evening with minced beef. Water in the aquaria was renewed daily with dechlorinated water adjusted to laboratory temperature.

Chemicals-Unless otherwise specified, all chemi ­cals used were of analytical grade and were obtained fro m Sigma Chemical Co., St. Louis, U.S.A. and Brit­ish Drug House Laboratory, Mumbai, India.

Induction and in vivo labelling of female-specific plasma proteins -Female murre ls were collected during prespawning period and injected im every day with 10 J1.g estradiol-17~ (E2)/100 g fish body weight fo r 7 days3. Twenty four hours prior to bleeding each fish was administered, ip, 0.25 mCi of 32P-NaH2P04 (sp. acti vity 104 mCi/mM; BARC, Mumbai). Fifteen minutes before collecti on of blood each fi sh was

SEHGAL & GOSWA MI : FE MALE-S PEC IFIC PLAS MA PROTE INS OF CHANNA PUNCTATUS 289

injected with 4-5 TI U of aprotinin. Blood samples were collected, centri fuged and the pooled plasma was processed immedi ately to prevent possible aggrega­ti on of pl as ma proteins 15. Pl asma was either subjected direc tl y to gel fil tration chromatography or after prior ultracentri fugat ion at 136,000 g for I hr to remove low-density li poproteins.

Isolation of plasma proteins-U lt rogel AcA 34 (LKB) packed in a glass colu mn (lOOx2.5 cm) was eq uili brated with several vo lumes of O. IM KCI, O. IM Tris-CI buffe r at p H 7.5 conta ining 0.2% sod ium azide ls. Plasma proteins were el uted at 4°C at a fl ow rate of 20 mllhr. Fractions of 5 ml were coll ected and the absorbance recorded at 280 nm. One hundred J1 1 from each fract ion was applied to Whatman 3 MM filter paper di sks and processed fo r radi oacti vity measurement 15 .

Preparation of antibodies-· New Zealand white rabbits were injected im with an emulsion of an equal vol ume of estrogenized murrel pl asma and Freund 's Complete Adjuvant four times at weekly intervals. Rabbits showing a positive response were admini s­tered a booster injection and bled one week later. Blood was allowed to clot at 4°C overnight and the serum stored in small aliquots at -20°C after addi tion of sodium azide. This anti serum is referred to as poly-

specific anti serum (PS AS). Two ml of thi s ant iserum was absorbed with 1.5 ml of normal male serum. The resultant anti serum showed no cross-reaction with male serum when tested by double immunodi ffus ion and is referred to as fe male-spec ific anti serum (FSAS) .

Imlllunolog ical analysis and autoradiography-32 P-labelled murrel plasma as well as fractions ob­ta ined after gel filtrati on were tested against PSAS and FSAS. Ouchterlony immunodi ffusion and immu­noelectrophoresis were perfo rmed fo llowing routine procedures. After development of precipiti n lines, the plates were washed free of excess reac tants and dri ed. They were coated with a thin film of photographic emulsion (Kodak NTB 3) and after seven days devel­oped in Kodak D-19b developer.

SDS PAGE and peptide mapping-Samples were subjected to polyacryl amide gel electrophoresis under denaturing conditions (SOS PAGE) on 5% gel. Pep­tide mapping of proteolytic digests was performed according to the procedure of Cleveland et af. 16. Sam­ples were digested with either papain or chymotrypsin at 37°C fo r 30 min and proteolysis stopped by heating the mixture to lOO°C for 2 min. The digests were electrophoresed at 200 V and stained with Coomass ie Brilliant Blue.

1.25 --------------.-------------.---- 25

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379

Fig. I -Gel filtration profile of .l2p labe lled plas ma from estrad io l-Irea ted murre l C. puncta/us. Almost the entire 32 p ac ti vity is associ­ated with vite llogenin (peak III ).

290 IND IAN J EXP BIOl, MARCH 2002

Results Elution profile and ill1l11unological identification of

plasma proteins in murre/-Figure I depicts the elu-

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tion pattern of 32P-Iabelled plasma of female murrel on Ultrogel AcA 34 after 7 injections of E2. Plasma re­solved into five absorbance peaks. The first peak,

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25 27 29 31 P

,----... 27 41 P 43 45

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41 53 P 55 57

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Fig. 2-0uchterlony double diffus ion in agarose gel. Alternate troughs contained 10011101' female- specific anti seru m (FSAS) and poly­specific antiserum (PSAS ). Wells conta ined alternate frac ti ons from the en tire ge l filtration profile depicted in Fig. I. The well numbers correspond with fraction numbers in Fig. I. Well P contained native plasma from E2-treated murreL Note a reac tion of non-identity be­tween fractions 27 and 39. Also note that all fractions of peak III and fractions of ascending part of peak IV in Fig. I reac ted with FSAS.

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Fig. 3-Gel filtration profile of 32 p labelled plasma from estradiol-treated murrel C. pUllctatus. 32p labelled plasma was ultracentrifuged at 136,000 g. The sub natant was chromatographed on UJtrogel AcA 34. Note the virtual absence of low density lipoproteins in the frac­tions in the void volume.

'1'

, -

SEHGAL & GOSWAMI: FEMALE-SPECIFIC PLASMA PROTEINS OF CHANNA PUNCTATUS 291

which eluted within void volume, was opalescent due to presence of low density lipoproteins with some 32p activity. Almost the entire 32p activity was associated with third peak (fraction s 23-33). This protein was characterised as vitellogenin on the basis of 32p activ­ity and its elution position (VeIVo 1.42). Interest­ingly, an additional 32P-containing component was observed at 1.8 elution position in the fractions (38-44) of the fourth peak.

Alternate fractions from the entire gel filtration profile were tested for immunoreactivity against PSAS and FSAS. The results presented in Fig. 2 show that with the exception of low density lipoprotein fraction (II), all other fractions crossreacted with PSAS. With FSAS, the precipitin reaction was con­fined only to fractions of third and ascending part of the fourth absorbancy peaks (see Fig. 2). A perusal of Fig. 2 clearly shows that the antigen in the IIIrd peak (fractions 23-33) was immunologically distinct from

that in the ascending part of IVth peak (fracrions 35c

41). Descending fractions (43, 45) of th IVth a showed no reaction with FSAS. When fraction 27 (111 peak) and fraction 39 (from IV peak) were placed in the adjacent wells, the precipitin arcs formed against FSAS exhibited a cross over reaction of non-identity (Fig. 2). Subsequently in the text, these two immu­nologically discrete female-specific plasma proteins are referred to as FS I and FS II respectively.

One step gel filtration chromatography on Ultrogel AcA 34 is sufficient to resolve murrel plasma pro­teins l5 . The present investigation showed that prior ultracentrifugation of 32P-labelled plasma at 136,000 g for I hr that removes low density lipoproteins, results in a better resolution of FS I and FS II. The gel filtra­tion profile of the clear subnatant is shown in Fig. 3. Almost the entire 32p was confined to FS I fractions. In contrast, FS II has no 32p activity. A small peak of radioactivity was associated with a protein , which

Fig. 4-0uchterlony double diffusion in agarose gel followed by autoradiography of 32p labelled FS I and FS II, and native plasma from estradiol-treated murre!. The central well contained FSAS. The peripheral wells A and 0 contained purified FS II, wells 8 and E con­tained purified FS I and wells C and F contained native plasma from estradiol-treated murrel (Fig. 4A). The corresponding autoradio­graph is depicted in Fig. 48. Note cross-over of precipitin lines of FS I and FS II and the confluence of precipitin lines in the plasma from estrogenized murre!. Note also that 32p radioactivity is exclusively associated with FS I (Fig. 48). Fig. 5- Immunoelectrophoresis of 32P-labelled plasma from estrogenized murrel and 32P-Iabelled purified FS I (Fig. SA) and FS II (Fig. 58). In both figures, well A contained 32P-Iabelled plasma (5 Ill) and trough contained 50 III of FSAS. Well 8 contained identical amount of purified FS I (Fig. SA) and purified FS II (Fig. 58). Note that un fractionated plasma of estrogenized murrel revealed only two female­specific proleins with different electrophoretic mobilities corresponding to purificd FS I (Fig. SA) and FS II (Fig. 58).

INDIAN J £XP BIOL, MARUI 2002

Fig. 6-0uchterlony double d iffusion in agarose ge l. The centra l we ll contained 50 11 1 female-specific aIlliserum (FSAS). Well s A and D conta ined purifi ed FS II ; well s B and E contained purified FS I in F igs 6A & 6B; wells C and F conta ined plasma from nor­mal male murrel (Fig. 6A) o r plas ma from normal vite ll ogeni c female murre l (Fig. 6B). In Fig. 6C wel ls A and D contai ned puri­fied FS II and FS I respectively, wells B, C, E and F conta ined

crude homogenate (10 11 1) of ovary from grav id murre l. Note the abse nce of precipitin line aga inst male plasma (Fig. 6A) and pres­ence of two precipitin lines against vitellogen ic fe ma le plasma (Fig. 6B) as well as aga inst ovarian homogenate (Fig. 6C). A lso note the cross-over (reaction of non-identity) between precipitin lines FS I and FS II (Figs 6A & 6B). ConOuence of ei ther li ne (reac ti on of identity) with precipitin lines agains t vi te lloge nic fema le plasma (Fig. 6B) and against ovari an ho mogenate (Fig. 6C) .

eluted after FS II. These two proteins (FS I and FS II ) iso lated by gel filtrat ion chromatography of ultracen­trifuged pl as ma of est rogeni zed murrel were em­ployed for a more detail ed immunological characteri­zation .

Resu Its of rad ial i mmunodi ffusion ex peri n.ents show that FS I and FS II are immunologically distinct and both proteins are present in the unfractionated E]­treated plasma. When Ouchterlony plates were sub­jected to autorad iography , the radioacti vity was found to be associated onl y with FS I; radioactive precipitin line corresponding to FS If was completely absent both in the nat ive pl asma as well as in the isolated protein (Fig. 4). When plasma was subjected to im­munoelectrophoresis, two precipitin arcs wi th differ­ent electrophoretic mobi lity were formed against FSAS; of these, one corresponded to purified FS [ and the other to puri fied FS II (Fig. 5) .

Immunoelectrophoresis foll owed by au torad iogra­phy also confirmed the absence of 32p rad ioactivity in FS II. Plasma fro m normal untreated male murrel showed no reaction with FSAS (Fig. 6A), whereas the abc - a - b -

, A

.. --

o

.. ~ , F ® ~- . ....-..----Fig. 7-Electrophoretic pattern on SDS PAG E (Fig. 7A) and Pept ide maps (Fig. 7B) of FS I ( lane a) and FS Il (l ane b). Marker prote ins ( lane c ill Fig. 7 A) e lec trophoresed were A: myosin (205

kD), B: ~-ga l aetosidase ( 11 6 kD), C: phosphory lase (97 kD), D: bov ine serum albumin (66 kD) , E: chicken egg a lbu min (45 kD)., F: carbonic anhydrase (29 kD). Samples were loaded on 5% re­solving ge l. For pept ide maps (Fig. 7B) eit her protein at a final concentrati on of 0.5 mg/ml in sample buffer, was incubated at 37"C fo r 30 min with 5 I1g/ml of chymotrypsi n (subst rate : e n­

zy me:: IOO:I ). 25 I1g of subst rate was loaded per we ll of a 10% reso lvi ng ge l.

1

SEHGAL & GOSWAMI: FEMALE-SPECIFIC PLASMA PROTEINS OF CHANNA PUNCTATUS 293

native plasma from normal seasonally vitellogenic murrel formed two precipitin lines with FSAS which exhibited reactions of complete identity with the re­spective precipitin lines of FS I and FS II (Fig. 68). Further, these two female-specific proteins were also present in ovarian homogenate of gravid murrel, since of the two precipitin lines formed between FSAS and the ovarian homogenate, one precipitin line was con­fluent with purified FS I and the other with purified FS II (Fig. 6C).

SDS PAGE and peptide mapping-On SDS PAGE two proteins, FS I and FS II, resolved into one peptide (Fig. 7 A). The molecular weight of FS I peptide was 175kD whereas that of FS II was 110 kD. Papain and chymotrypsin were used for digestion. Papain did not generate fragments of sufficiently wide range of mo­lecular weights. However, a satisfactory pattern was observed with chymotrypsin. The peptide maps of the two proteins, FS I and FS II, were completely differ­ent, with each protein containing numerous peptides of the mobility not represented in the other (Fig. 78).

Discussion The present study clearly demonstrates the pres­

ence of two distinct female-specific plasma proteins in the plasma of female murrel, C. punctatus, follow­ing exogenous administration of estradiol-] 7~. Both the proteins are absent in the plasma of male murrel3. Out of the two, only one (FS I) contains alkali-labile phosphorus and can incorporate radioactive phospho­rus, thereby indicating that this protein is vitellogenin. In murrel, FS II is a unique non-phosphorylated plasma protein whose chromatographic and electro­phoretic behaviour differs from FS I (vitellogenin).

The elution pattern of E2 plasma on UJtrogel AcA 34 shows that FS II is sometimes insufficiently sepa­rated from other plasma components. Prior removal of lipoproteins by ultracentrifugation greatly improves the resolution of FS II from vitellogenin and from other plasmatic proteins. This protein lacks radio­labelled phosphorus.

A minor phosphoprotein (elution position 1.8) is observed in the plasma of normal untreated male mur­re!. Similar protein is reported in the plasma of Er treated male and that of vitellogenic female murre!. Neither the concentration nor the 32p activity of this component changes significantly after E2 treatment3. Presence of this protein in either sex at any time of the year suggests that it is not a sex-specific phosphopro­tein.

Unfractionated (native) plasma from normal sea­sonally vitellogenic female murrel as well as from Er treated murrel yields two distinct precipitin lines against FSAS; one of these is confluent with the pre­cipitin line formed against purified vitellogenin (FS I) and the other with that formed by FS II. This shows that FS II is not an arti fact but a natural plasma pro­tein of vitellogenic murre!' Further, two precipitin lines are formed against FSAS by a crude homogenate of yolky oocytes, of which one shows reaction of identity with vitellogenin and the other with FS ]1. The immunological similarity between vitellogenin and egg-yolk proteins is only to be expected, since the former is an established precursor of the latter. That vitellogenin shares antigenic sites with egg-yolk pro­teins has often been demonstrated8. 9.

The present results further show that the two fe­male-specific plasma proteins of murrel are immu­nologically different from each other. This is evi­denced by the fact that these two proteins exhibit pre­cipitin reactions of complete non-identity with FSAS. Further, the two proteins have entirely different elec­trophoretic mobilities, FS I (vitellogenin) being more negatively charged than FS II . The molecular weight of FS I peptide is more than that of FS II peptide. Fur­ther, autoradiography shows that unlike vitellogenin , FS II lacks phosphorus. Peptide mapping is a stringent test of protein identity and that if two peptide profiles are different then the original proteins are not closely related. The highly divergent peptide profiles of FS I (vitellogenin) and FS II observed in the present study should, therefore, be regarded as sufficient proof that the two proteins are dissimilar and unrelated.

Estrogen stimulates not only the synthesis of vitel­logenin but of a variety of other components which, like vitellogenin, are eventually deposited in the 00-

cytes2.5.8.9.13.14. For example, in many avian and fish species estradiol promotes, in addition to vitellogenin synthesis, a pronounced accumulation of low and very low density lipoproteins which are sequestered by the

. 17·19 E I' d h grow Ing oocytes . strogens a so In uce t e syn-thesis of several vitamin-carrying proteins, specially those concerned with the binding and transport of ri­boflavin and thiamine which are considered essential for embryonic development2.8.9. Polymorphic forms of estrogen-dependent proteins have been reported in the plasma and egg-yolk of quail, Coturnix coturnix2o. In Xenopus, Holland and Wangh21 have reported an additional protein (EP 45) which is responsive to estradiol treatment but is absent in normal males. Yet another protein (EP 20) is present in both sexes of

294 INDIAN J EXP BIOL. MARCH 2002

Xenopus but its levels increase four-fold following estradiol treatment21.

In majority of fi shes vitellogenin has been reported to be the only female-specific plasma protein . None­theless, in plasma of few fishes, precursor for egg en­velope proteins, a female-specific protein other than . II . I b d ,o. ' 4 JJ·J8 I h vlte ogenlll, las een reporte .-- - . n co 0

salmon, Oncorhynchus kisluch, three egg-yolk com­ponents are present in the plasma of sexually matur­ing and estrogenized female fish 22 . Two egg-yolk components, EI and E2, of the rainbow trout exhibit an immunological reaction of non-identity when tested against a female specific antiserum; however, vitellogenin spurs over either protein suggesting that these two egg-yolk proteins are derived from vitel­logenin29. A significant finding reports on the isola­tion from the ascites fluid of medaka, Oryzias lalipes, of spawning female-specific protein other than vitel­logenin which cross reacts with an antichorion glyco­protein antibody,o.'3.28 Hyllner et al .25 and Hyllner and Haux30 have detected plasma proteins, immunologi­cally related to vitelline envelope proteins, which can be induced by estradiol administration in both sexes in rainbow trout (Oncorhynchus mykiss) , brown trout (Salmo trutta) and turbot (Scophthalamus maximus). Similar observation has been reported by Larson et at. 27 . Molecular biological analysis of egg envelope has suggested that vitelline envelope protein genes are expressed in the liver only, implying an extra-ovarian origin of these proteins 14.3 1. These results suggest that some major components of egg envelope" be synthe­sized in the liver under the influence of estrogen.

Egg yolk provides nutrition whereas egg envelope protects the developing embryo. Vitellogenin and chori ogenin, precursor proteins for egg yolk and egg envelope respectively, are synthesized in the liver un­der the influence of estradiol-17~. The present study shows that two unrelated proteins, FS I (vitellogenin) and FS II , are synthesized by the hepatocyte under the influence of estradiol-17~. Both the proteins are transported via blood to the ovaries where they are incorporated in the oocytes.

Acknowledgement

This work was supported in part by Research Grants from the Department of Science and Techno­logy, New Delhi and from the leAR, New Delhi.

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..

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SEHGAL & GOSWAMI: FEMALE-SPECIFIC PLASMA PROTEINS OF CHANNA PUNCTATUS 295

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