Further biochemical studies on carp gonadotropin (c-GTH); Biochemical and biological comparison of c-GTH and a gonadotropin from Acipenser stellatus pall. (Chondrostei)

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

29, 498-505 (1976)

Further Biochemical Studies on Carp Gonadotropin (c-GTH); Biochemical and Biological Comparison of c-GTH and a Gonadotropin from Acipenser stellatus Pall. (Chondrostei)’ E. BURZAWA-GERARD, B. GONCHAROV,~ A. DUMAS AND Y .A. FONTAINE Laboratoire de Physiologie d’Endocrinologie cornparke

du Mu&urn national associe’ au C.N.R.S.,

d’Histoire naturelle; Laboratoire 7, rue Cuvier, Paris 5e, France

Accepted March 1, 1976

Our knowledge of fish gonadotropins (GTH) is still fragmentary. Highly purified carp GTH (c-GTH) was obtained by ion exchange chromatography, gel filtraand preparative electrophoresis tion (Burzawa-Gerard, 1971). The hormone from a Pacific salmon, Oncorhynchus tshawytscha, was purified by ion exchange filtration chromatography and gel (Donaldson et al., 1972). In both cases only one gonadotropin was apparently present, of which a number of biochemical and biological properties have been described 1965; (Burzawa-Gerard and Fontaine, Donaldson et al., 1972; Burzawa-Gerard, 1974a). However, further work is needed to gain a deeper insight into the structure of these fish hormones. On the other hand, more piscine species, especially nonteleosts, have to be investigated. ’ Work supported in part by D.G.R.S.T. (Biologie de la reproduction et du developpement) and C.E.A. (Department of Biology). This paper represents a contribution at the 7th International Symposium on Comparative Endocrinology (July 1974). Therefore it was focused on our own results, without extensive discussion. Its publication has been unfortunately delayed because of accident of transmission from the Symposium organizers to the editors of this journal. * Present address: Institute of Developmental Biology, Academy of Sciences of USSR; 26 St. Vavilov, Moscow 117 334, USSR.

We report here recent progress these two lines of research. I.

@ 1976 by kcademic Press, Inc. of reproduction in any form reserved.

AND METHODS

Starting material. c-GTH was purified as previously described (Burzawa-Gerard, 1971) from an acetonic powder obtained from the Staller Fisheries (Spirit Lake, Iowa). Sturgeon pituitaries were collected in the USSR, during the upstream reproductive migration in the Volga, from mature animals of both sexes. An acetonic powder was then prepared. Bioassays. The frog spermiation test was carried out as previously described by Fontaine and Chauvel (1961). The occyte maturation in vitro was performed on several Amphibians (Bufo bufo, Bufo calamita, Rana temporaria) and on the Sturgeon (Acipenser stellatus); this method is described elsewhere (see Burzawa-Gerard, Goncharov and Fontaine, 1975). The median effective dose (DsO) and/or potency ratios were calculated, with the limits for P = 0.05, according to Emmens (1948). The adenyl cyclase activity in homogenates of goldfish ovaries was measured as previously described (Salmon et al. 1974). Dissociation of GTH’s. The gonadotropins were dissociated either by molar propionic acid or urea, 8 M, at room temperature (protein concentration: 2-10 mg/ml). Incubations were first carried out for 12 to 24 hr but it was later observed that shorter incubations were as efficient. Reassociation of c-GTH. The subunits were mixed in the molecular weights ratio (11,000/19,ooO) and dissolved in phosphate buffer 0.02 M, pH 7.0) with a total protein concentration of 10 mg/ml at room temperature (20-23°C). They were incubated for 15 min or 5 hr. The solution was them diluted with gelatine buffer ( l”lm., pH 7.0) for bioassays.

498 Copyright All rights

MATERIAL

along

499

FISH GONADOTROPINS

Such a separation was indeed carried out column of diethylaminoethyl i:phaadex A 50 (Burzawa-Gerard, 1974b). Figure 1 shows that the elution pattern for the urea-treated hormone was very different from that obtained for the native hormone. One unadsorbed peak appeared and the second main fraction, which had been adsorbed, was eluted for an ionic strength higher than in the case of the native c-GTH. The unadsorbed protein will be provisionally called SU I and the absorbed one SU II. Some of their properties are summarized in Tables 2 and 3. It should be noticed that SU I is likely to be smaller than SU II. Both were much less active on frog spermiation than c-GTH. Preliminary experiments on goldfish ovarian adenyl cycII. QUATERNARY STRUCTURE OF lase indicated that SU I was at least 1000 c-GTH times less active than c-GTH. The low but After treatment by molar propionic acid significant activity of SU II (around l/20 of the biological activity of c-GTH was that of c-GTH) may reflect a contamination strongly lowered and the molecular weight by undissociated hormone; indeed SU II was about half the initial value (Table 1). and c-GTH would not be completely sepaThese results indicate that c-GTH dis- rated on diethylaminoethyl Sephadex (see sociates into subunits under the influence of Fig. 1). molar propionic acid. Such a dissociation When SU I and SU II were incubated was also observed in urea, 8 M. Analytical together, even for a short period of time, an electrophoresis on polyacrylamide gel important biological activity was again presconfirmed this conclusion and also showed ent (Table 3). that the two subunits, which had very difIn analytical electrophoresis on polyacferent Rf values, could probably be sepa- rylamide gels at pH 8.9 SU I did not mirated by ion exchange chromatography. grate, whereas the Rf of SU II and c-GTH Purification methods. Gel filtrations on Sephadex G 100 were performed as described by Flodin and Killander (1%2). Ion exchange chromatographies on diethylaminoethyl cellulose were carried out according to Sober et 01. (1956); the adsorbed material was eluted by a gradient of ionic strength. The chromatography on diethylaminoethyl Sephadex A 50 was performed in a similar way. Analytical methods. The sedimentation rate (S2,& was determined by ultracentrifugation in sucrose gradient as described by Fontaine and Condliffe (1964). The centrifugation was carried out, at 65,000 rpm/min in a Spinco 65, either in a phosphate buffer (pH 7.4) NaCl, 0.1 M, or in molar propionic acid. Disc-electrophoresis on 7.5% polyacrylamide gel was performed at pH 8.9 as described by Davis (1964). Electrophoresis in presence of urea 8 M was done in 7% acrylamide gel; it was necessary to deionize urea by chromatography on a mix bed ion exchanger (AG 501-X 8 D).

REVERSIBLE

Method Gel filtration Sephadex G 100 Ultracentrifugation Bioassay on frog (Ram esculenta) (spermiation)

DISSOCIATION

TABLE 1 c-GTH BY MOLAR

OF

PROPIONIC

ACID”

Propionic acid + lyophylisation

Constant

c-GTH

Propionic acid .O M

&I

0.25-0.28

0.41-0.43

0.25-0.28

2.5-2.6 27,000-28,000

1.65 14,000-15,000

2.5-2.6 27,000-28,000

S 20.w MW D,, (&animal)

a Effects on molecular size and biological activity.

2.5 (1.8-3.4)

20 (13-30)

3.7 (2.0-9.4)

500

ET AL.

BURZAWA-GERARD

60

I

100

160

200 15

$11

I

1

IIII

-0.2

- 10

5

/ FIG. 1. Separation of the subunits of c-GTH: chromatography on diethylaminoethyl Sephadex A 50 of native c-GTH (A) and 8 M urea-treated c-GTH (B). The column (2 x 22 cm) was equilibrated in Tris-HCl buffer (0.05 M, pH 7.7). The elution was carried out by a gradient of ionic strength (Tris-HCl buffer, 200 ml-NaCl, 0.6 M, 200 ml). Fractions-of 3 ml were collected. (0) Proteins (O.D. 276 nm); (A) conductivity (mMH0).

TABLE MOLECULAR

SIZE AND ELECTROPHORETIC

Gel filtration Sephadex GlOO (NH,HCO,, 0.1)

su I su I1

2 BEHAVIOUROF

c-GTH SUBUNITS Electrophoresis on polyacrylamide gel (PH 8.9)

Ultracentrifugation on sucrose gradient

KLI

s 2o.w

MW

Rf

0.42 0.38

1.4 2.0

11,100 19,ooO

No migration 0.78&.80

FISH

501

GONADOTROPIN!3

TABLE 3 OF c-GTH SUBUNITS~

REASSOCIATION

SU I + SU II; incubation: Native c-GTH

su I

su II

15 min

5 hr

2.8” (1.3-6.8)c

>50*

2506

5.0* (2.6-9.5)c

6.6* (5.3-8.2)c

a Effects on their biological activity. * DsOon frog spermiation ~gkmimal). c Limits for P = 0.05.

were, respectively, 0.78-0.80 and 0.480.51. After incubation of SU I + SU II, a new band was again observed, which had the same Rf as c-GTH (Fig. 2). In conclusion, c-GTH is made of two dissimilar subunits which have been separated. Moreover, these two subunits are able to reassociate in a molecule very similar to the native hormone. III. THE GONADOTROPIN (aci-GTH) FROM THE STURGEON (Acipenser stellatus Pall.) AND ITS COMPARISON WITH c-GTH A.

Purification

of aci-GTH

Preliminary experiments showed that aci-GTH was less acidic and less stable at high ionic strength than c-GTH. Therefore some steps of the procedure previously used for the carp hormone had to be changed. For instance diethylaminoethyl

1

2

3

4

FIG. 2. The c-GTH subunits and their reassociation: electrophoretic study. (1) Native c-GTH; (2) SU I; (3) SU II; (4) SU I + SU II previously incubated in phosphate buffer (pH 7.0, 0.02 M). The gels were stained with 1% Amidoschwartz in 7% acetic acid solution.

cellulose chromatography was carried out at pH 9.4 instead of 7.7 and the hormonal solutions were concentrated by dialysis under vacuum instead of flash evaporation. The purification procedure, which is described elsewhere in detail (BurzawaGerard, Goncharov ‘and Fontaine, 1975a) included saline or alcoholic extraction, gel filtrations and diethylaminoethyl cellulose chromatography of which a typical elution pattern is represented in Fig. 3. In various experiments, biological activity was determined both on frog spermiation (in vivo) and on the maturation of Bufo oocytes (in vitro); there was no significant discrepancy between the two methods. No significant biological activity was detected in the unadsorbed fraction. The adsorbed, biologically active, fracwas concentrated, filtered on tion Sephadex G 100 in a volatile buffer and then lyophilized; the resulting material was called aci-GTH. About 2 pg of this material induced the spermiation in 50% of the injected frogs. The biological activity of aciGTH was also compared to that of the starting material on several tests (Table 4). The small differences between the various purification ratios were easily accounted for by the different times at which the assays were made and by the different preparation of pituitary powder which were used. Indeed, there was no significant indication of the selective separation of a principle more active in one test than in the others. Because of the general lack of specificity of responses of amphibian gonads (BurzawaGerard and Fontaine, 1972), these results

502

BURZAWA-GERARD

ET AL.

a a276(Bjt)

.3 as-2

-1

a l*

2b

3im

&Ill

4&l

861~ Efllua~t(IRI)

7&

FIG. 3. Purification of aci-GTH: chromatography on diethylaminoethyl cellulose of the active fraction obtained by Sephadex G 100 gel filtration of an extract of sturgeon pituitary. The column (2 x 20 cm) was equilibrated in sodium glycinate buffer (0.02 M, pH 9.4). The elution was carried out by a gradient of ionic strength (sodium glycinate buffer (0.02 M, pH 9.4, 225 ml) (sodium glycinate buffer (0.5 M, pH 9.4, 225 ml). Fractions of 9 ml were collected. (O-) Proteins (O.D. 276 nm); conductivity (m MHO); (C) biological activity on frog spermiation expressed in terms of aci-GTH (4.38 A).

suggest that all the gonadotropic principles possibly present in Acipenser pituitary were purified together and present in aciGTH. B.

Properties

of aci-GTH and Comparison with c-GTH 1. Biochemical Properties Analytical electrophoresis of aci-GTH on acrylamide gel at pH 8.9 showed the pres-

PREPARATION

OF

Rana

esculenta

Rana

temporaria

Spermiation in vivo Maturation

of oocytes

in vitro

Maturation

bufo

Acipenser

TABLE 4 aci-GTH: PURIFICATION RATIO ON VARIOUS Tests

Species

Bufo

ence of two main bands (a and b) and a smaller one (c) of Rf 0.27, 0.31 and 0.35, respectively. Preliminary experiments indicated that a significant biological activity was only found in a and b. aci-GTH appeared therefore less acidic than c-GTH. The sedimentation rate of aci-GTH (S,,, = 2.4-2.8) and its behavior on Sephadex G 100 (K, = 0.30-0.31) were close to the values for c-GTH.

of oocytes

in vitro sfellatus

Maturation

of oocytes

in vitro

SPECIES AND

TESTY

Ratio 10.1 ( 5.8-17.9) 14.0

(13.4-14.7) 12.1 (10.7-13.4) 11.0 ( 9.9-12.9)

FISH

DISSOCIATION

OF

TABLE 5 aci-GTH BY MOLAR PROPIONIC

Sedimentation rate (S 20,W)

2.4-2.8

ACID

AND

UREA”

Activity on frog spermiation (Ds,, &animal)

Propionic acid, l.OM

pH 1.4

503

GONADOTROPIN!4

pH 7.0

1.5

pH 8.0

(LEO)

(l.Z.2)

Propionic acid, 1.O M

Urea, 8M

220

220

a Effects on molecular size and biological activity.

1

2

3

Dissociating agents led to changes similar to those observed in the case of the carp hormone; both sedimentation rate and biological activity were decreased (Table 5). Urea-treated aci-GTH was also studied by analytical electrophoresis (Fig. 4). The bands corresponding to aci-GTH were still visible but a new protein zone was present, with two main bands of R, 0.53 and 0.59. Further work which is described elsewhere (Burzawa-Gerard, Goncharow and Fontaine, 1975b) allowed us to isolate from aci-GTH the fraction corresponding to the “a” band. This fraction, when treated by urea, also gave rise to two bands with Rf of 0.53 and 0.59. These results indicate that the sturgeon hormone, like the c-GTH, is

4

FIG. 4. aci-GTH and its dissociation by urea: electrophoretic study. (1) aci-GTH (4.38 A); (2) aciGTH (4.38 A) + standard protein (R, = 0.85); (3) aci-GTH (4.38 A) previously incubated in 8 M urea, 1 hr in Tris-phosphate buffer (pH 7.2) (2 mg/ml at room temperature). (4) aci-GTH (4.38 A) + standard protein (R, = 0.85) previously incubated in 8 M urea, 1 hr in Tris-phosphate buffer (pH 7.2) (2 mg/ml) at room temperature. The gels were stained with 1% AmidoSchwartz in 7% acetic acid solution.

TABLE COMPARISON

OF THE BIOLOGICAL

Species

ACTIVITIES

OF

6 aci-GTH AND c-GTH

Tests

ON SEVERAL

TESTS

Activity ratio

aci-GTH c-GTH

Rana temporaria

Maturation of oocytes in vitro

13.7 (12.4-14.9)O

Rana esculenta

Spermiation in vivo

2.6 ( 1.7- 3.6)“

Bufo

bufo

Carassius aura&s n Limits for p = 0.05.

Maturation of oocytes in vitro

27.7 (25.1-30.4)=

Stimulation of ovarian adenyl-cyclase in vitro

# 0.001

504

BURZAWA-GERARD c AlP accunrl~tion

ET AL.

(P moles/min) .

131211 -

-

CGTH

C--.

aci6TH

.

10 -

0

.

9a-l-

/-

.

6---i-

5.. 4-

8 o

.

n _____._._____

l.

_____-

----I---------

. .

0

, 1

10

100

1000

Hormonal concentration (/g/ml

)

FIG. 5. Comparison of the effects of c-GTH and aci-GTH on the ovarian adenyl cyclase from immature goldfish. The incubation was carried out at 20°C for 10 min. The composition of the incubation medium was: HCI-Tris buffer (pH 7.4), 0.020 M; phosphoenol pyruvate, 0.0072 M; pyruvate kinase, 12 units/ml; 32P-a ATP, 0.002 M and ~30 cpm/pmole; MgCI,, 0.006 M; cyclic AMP, 0.002 M; bovine serum albumin, 1 mgiml; sucrose. 0.1 M.

made of two subunits. However, preliminary results suggest that the characteristics of the equilibrium between the hormone and the subunits are not identical for aciGTH and c-GTH. 2. Biological Properties The activity of aci-GTH was determined by several bioassays, relative to c-GTH. aci-GTH is slightly more potent than c-GTH on frog spermiation but it is about 1000 times less active on goldfish adenyl cyclase (Fig. 5). Finally aci-GTH is lo-30 times more active than c-GTH on the maturation of amphibian oocytes (Table 6). These results show that an important zoological specificity of action of the gonadotropins can exist even within fish, a conclusion which is in agreement with previous works (Clemens and Johnson, 1965; Fontaine et al., 1972; Breton et al., 1973). Finally, the fact that sturgeon GTH is more active than carp GTH on Amphibian suggests that aci-GTH is closer to amphibian hormones than is c-GTH. More biochemical work is needed to test this hypothesis.

REFERENCES Breton, B., Billard, R., and Jalabert, B. (1973). Specificitt d’action et relations immunologiques des hormones gonadotropes de quelques T&OSteens. Ann. Biol. Anim. Bioch. Biophys., 13 (3), 347-362. Burzawa-Gerard, E. (1971). Purification dune hormone gonadotrope hypophysaire de Poisson Teleosteen, la Carpe (Cyprinus carpio). Biochimie, 53, 545-552. Burzawa-Gerard , E. (1974a). Etude biologique et biochimique de l’hormone gonadotrope d’un Poisson Teleosteen, la Carpe (Cyprinus carpjo L. ). M&m. Mus. nat. His. Nat., T LXXXVI, p. l-77. Burzawa-Gerard, E. (1974b). Separation et reassociation des sous-unites de l’hormone gonadotrope d’un Poisson TClCosteen, la Carpe (Cyprinus carpio L). C. R. Acad. Sci., 279, 1681-1684. Burzawa-Gerard, E., and Fontaine, Y. A. (1%5). Activites biologiques d’un facteur hypophysaire gonadotrope purifie de Poisson Teleosteen. Gen. Comp.

Endocrinol.

5, 87-95.

Burzawa-Gerard, E., and Fontaine, Y. A. (1972). The gonadotropins of lower vertebrates. Gen. Camp. Endocrinol.

Suppl.

3, 715-728.

Burzawa-Gerard, E., Goncharov, B. F., and Fontaine, Y. A. (1975a). L’hormone gonadotrope hypophysaire d’un Poisson Chondrosteen, l’Esturgeon (Acipenser stellutus Pall.). I. Purification. Gen.

Comp.

Burzawa-Gerard,

Endocrinol.

27, 289-295.

E., Goncharov,

B. F., and Fon-

FISH

GONADOTROPINS

tame, Y. A. (1975b). L’hormone gonadotrope hypophysaire d’un Poisson Chondrosteen, I’Esturgeon (Acipenser stellarus Pall.). II. Proprietes biochimiques. Gen. Comp. Endocrinol. 27, 296304. Clemens, H. P., and Johnson, W. W. (1965). Specificity of the gonadal hydration factor in the pituitary of some fresh water fishes. Copeia 2, 389-398. Davis, B. S. (1964). Disq-electrophoresis. II. Method and application to human serum protein. Ann. N.Y.

Acad.

Sci.

121, 434427.

Donaldson, E. M., Yamazaki, F., Dye, H. M., and Philleo, W. W. (1972). Preparation of gonadotropin from Salmon (Oncorhynchus tschawytscha) pituitary glands. Gen. Comp. Endocrinol, 18, 469-48 1. Emmens, C. W. (1948). Principles of biological assay. Chapman and Hall Ltd., London. Flodin, P., and Killander, S. (1962). Fractionation of human serum protein by gel filtration. Biochem. Biophys. Acta 63, 403-tlO. Fontaine, M., and Chauvel, M. (1961). Evaluation du

505

pouvoir gonadotrope de l’hypophyse des Poissons Teleosteens, et en particulier du Salmo s&r L. a diverses &tapes de son developpement et de ses migrations. C. R. Acad. Sci. 252, 822-824. Fontaine, Y. A., and Condliffe, P. G. (1964). Density gradient centritirgation of bovine thyroid stimulating hormone. Biochemistry 2, 2X&253. Fontaine, Y. A., Salmon, C., Fontaine-Bertrand, E., Burzawa-Gerard, E., and Donaldson, E. M. (1972). Comparison of the activities of two fish gonadotropins on adenyl-cyclase. Canad. .I. Zool. 50, 1673-1676. Salmon, C., Delerue-Le Belle, N., and Fontaine, Y. A. (1974). Influence de la temperature sur I’adbnyl cyclase ovarienne et sa stimulation hormonale. Etude comparee chez le Rat et un Poisson Teleostien (Carassius auratus). Biochimie, 56, 1229-1238. Sober, H. A., Gutler, F. J., Wyckoff, M. M., and Peterson, E. A. (1956). Chromatography of proteins. II. Fractionation of serum proteins on anion exchange cellulose. J. Amer. Chem. Sot. 78, 756-763.