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Pituitary gonadotropic hormone from a chondrostean fish, starred sturgeon (Acipenser stellatus Pall.)
GENERAL
AND
Pituitary
COMPARATIVE
ENDOCRINOLOGY
49, 375-382 (1983)
onadotropic Hormone from Sturgeon (Acipenser s IV. Differences
B. F. GONCHAROV,
in Biological Action of Sex-specific Forms
A. A. KUZNETZOV,
AND E. BURZAWA-GERARD*
N. K. Koltrov Institute of Developmental Biology, USSR Academy of Sciences, Moscow 117 808, USSR, and *Laboratoire de Physiotogie g&&ale et comparie, d’Endocrinologie comparPe associ4 au CNRS, MusCum national d’ffistoire 7 rue Cwier, 75230 Paris Cedex 5, France
26 Vavilov St., L~boratoi,re narur-cl/u.
Accepted March 30: 1982 The specific activities of four subfractions of the starred sturgeon (Acipenser stellarus Pall.) pituitary gonadotropin (aci-GTH-A, -B, -C, -D) were compared using several test systems. The ratios of two of them (A and D) that contained the isoforms differing by the values of isoelectric point were shown to be different, especially when irz vitro oocyte maturation and spermiation tests were used. These differences were not due to the presence in one of these preparations of the component affecting spermiation rather than oocyte maturation. The qualitative differences in the biological action of aci-GTH-A and -D were also revealed by the comparison of dose-response curves using toad oocyte irr vitro maturation. Finally, as the spectra of aci-GTH isoforms were practically identical when female or male individual pituitary extracts were submitted to isoelectric focusing, we concluded that the structural and functional heterogeneity of aci-GTH was related neither to sex nor to genetic intrapopulation polymorphism.
It is now accepted that the polymorphism of vertebrate pituitary gonadotropic hormones may have a functional significance (see Bogdanove and Nansel, 1978; Wakabayashi, 1980). Interesting aspects of this problem have arisen when fish gonadotropins have been investigated. The existence of sex-specific forms of pituitary gonadotropic hormone has been suggested (Idler et ai., 1975; Breton et ai., 1978; Burlakov et ai., 1979). An attempt has also been made to elucidate the relationship between the relative amount of electrophoretically different gonadotropic fractions in the carp pituitary and the stage of ovarian development (Burlakov and Gureeva-Preobrazhenskaya, 1977). In the accompanying paper (Kuznetzov et al., 1983) we described the purification of four active subfractions of sturgeon pituitary gonadotropin (a>H) that differed by the relative content of differently charged
isoforms. The present work was done to study whether these differently isoforms were identical by their b properties. Two kinds of approaches were used to answer this question. First was the comparison of the ratio of the specific activities of the studied hormone preparations determined with different test systems. As the second approach, we used the comparison of the form of dose-response curves using amphibian oocyte in ~12ro rna~~rat~o~. In both cases the qualitative differences in the biological action of two aci-GTH subfractions extreme by isoelectric point (p1) values of their molecules were revealed. We also compared the GTH isoforms in the indiv of female and male fishes significant differences. MATERIALS
AND METHO
Four subfractions of aci-GTE (A,B.C.D; were studied in the present work. Their purification. Materials.
375 0016.6480/83/Q30375-08$0i.5o:o Copyright All rights
@ 1983 by Academic Press. inc. of reproduction in any form resewed
376
GONCHAROV,
KUZNETZOV,
separation, and some physical and chemical properties are described elsewhere (Kuznetzov et al., 1983). Individual pituitary glands were collected from sexually mature female and male sturgeons (Acipenser stellatlds Pall.) caught in the Volga river during their anadromous migration. After dissection pituitaries were either acetonized or frozen and kept at -20 until use. Bioassa~s. Amphibian and sturgeon oocyte in vitro maturation test (Thornton, 1971; Goncharov, 1971a, 1972a) was used as earlier described in detail (BurzawaGerard et al., 1975). Frog (Rarra tempornuia) spermiation test was realized in the conditions described elsewhere (Fontaine and Chauvel, 1961). In addition, two methods of registration of spermiation were used: qualitativewhen the presence or the absence of spermiation was registered, and semiquantitative-when the intensity of reaction was estimated and expressed in arbitrary units (1, weak reaction-single spermatozoa are in the sample: 2, middle reaction-not more than 10 spermatozoa are seen in the field of vision of microscope at x 100 magnification: 3, strong reaction-more than 10 spermatozoa are seen under the same conditions). Isoelectric focusing. Analytic polyacrylamide gel isoelectric focusing of the extracts of individual pituitaries followed by the determination of gonadotropic activity distribution using the toad (Bufo viridis) oocyte in vitro maturation test was carried out as de-
I
OOCYTE Acipenser steilotus
AND BURZAWA-GERARD scribed earlier (Kuznetzov et al., 1983). When the frog spermiation test was used to localize the gonadotropic activity after isoelectric focusing, gel was sliced in 3-mm fragments. Each fragment was then placed in a flacon containing 2.5 ml of amphibian Ringer solution. After the overnight elution at 4”, the OS-ml aliquots were injected into the male frogs. Stutistical methods. The median effective dose (ED,,) and/or potency ratios were calculated with limits for P = 0.05 according to Emmens (1948). Sign test was used according to Urbach (1964).
RESULTS
Comparison of specific activities of four aci-GTH subfractions using several test systems. In Fig. 1 are shown the relative potencies of four aci-GTH subfractions (A,B,C,D) as compared by their ED,,. According to in vitro oocyte maturation test (whatever species was used), three aciGTH preparations (A,B,C) were of about equal specific activity, while aci-GTH-D was 2-3 times less active. On the contrary, the frog spermiation test did not reveal any significant differences between all aciGTH subfractions whatever method of reg-
s PERMIATION
MATURATION Bufo vi ridis
RQIICI temporario
[email protected]
I
i 20
temporaria
FIG. 1. Comparison of specific activities of four subfractions of aci-GTH (A,B,C,D) determined by several bioassays. In the case of irz vitro oocyte maturation test the experiments have been repeated with follicles of two females of each species. 1, qualitative, and 2, semiquantitative method of registration of spermiation reaction (see Materials and Methods for details). Limits for P = 0.05.
POLYMORPHISM
SPECIFK
ACTIVITY
RATIO
Test reaction:
Species: 9 1 92 Note.
Calculations
OF
STURGEON
GONADOTROPIN.
TABLE aci-GTH-Naci-GTH-D In vitro
oocyte
1 DETERMINED
BY SEYER+L
Bro~ss~ys
maturation
Spermiation
Acipemer stellarus
Rar1a
ternporaria
3.1 (2.6-3.8) 2.9 (2.5-3.4) are done from
34?
IV
1.7 (1.5-2.0) 1.9 (1.7-2.2) the results
presented
1.7 (1.5-1.9) 2.1 (1.8-2.5) irr Fig.
1. I+, ?+ please
‘+l.l (W-3.4) Z-1.1 (0.9-1.3) see legend
to Fig.
i. Liinits
cob;
P = Q.05.
istration-qualitative or semiquantitative (see Materials and Methods) was used. To avoid the inaccuracy, the same preliminary frozen stock aci-GTH preparations solutions were used for the comparison of then- specific activities with R. temporaria oocyte maturation and spermiation tests. Thus, the significant discrepancy in the relative potency of aci-GTH-A and -D was with different test-systems used (Table I). Cornparisotz of the profiles of gonadotropic activity of aci-GTH-D submitted to isoelectric .focusing as determined by oocyte maturation and sperrniation test. The
observed discrepancy in the ratios of specific activities of aci-GTH-A and -D obtamed with oocyte maturation and spermiation tests could be explained by the fact that aci-GTHcontained a component which manifested itself only or predominantly in the spermiation test. However, when the eluates of gel fragments obtained after isoelectric focusing of aci-GTH-D were tested with toad oocyte in vitro maturation or frog spermiation, no component predominantly acting on spermiation was observed (Fig. 2). On the contrary, the isoforms of aci-GTH with higher pZ values were revealed by oocyte maturation rather than by spermiation test. But it seems to be due to the low-er sensitivity of the frog spermiation test (see Fig. 1) rather than to the characteristics of these components. Comparison of dose-response curves for aci-GTH-.4 and -D. This experiment was
carried out to try to reveal qualitative
dif-
ferences in the action of acion toad oocyte maturation. us (Goncharov, 1971b) sh ~~ysio~ogica~ state of large follicles varied markedly in the hibernat late-spawning amphibians. cles of some of them were exposed ii? 13r.o to the increasing co~ce~t~at~o~ of pituitary suspension, the percentage of oocytes with GVBD (germinal vesicle breakdowns raised, reached the maximal level ~d~ffer~~t fior different females) and then went The maximal percentage of matur cytes that can be obtained as w of the dose-response curve v spect to species from which taken. This model system was used to corn the dose-response relationships of two 100
50
0
75
SC
5.5
50
p:’
2. Profile of gonadotropic activity after polyacrylamide gel isoeiectric focusing of aci-GTil-D in the range of pH 3.5-9.5. Gonadotropic activity determined by two bioassays: 1 (0) Bufo viridi$ oocyte 1~ Iaitro maturation (50 pg of protein were applied to gel: 40-60 follicles were used for each point): and 2 ( Rana temporaria spermiation (250 pg of protein were applied to gel: 5 males were used for each point). Ordinate-percentage cf oocytes with GVB.D (germinal vesicle breakdown) or frog males responded by spermiation. FIG.
378
GONCHAROV,
KUZNETZOV,
aci-GTH subfractions (A and D). The follicles of four toad females were used for this kind of experiment. The results are presented in Fig. 3. As the specific activity of aci-GTH-A is approximately twice higher than that of aci-GTH-D, the dose scales are shifted correspondingly. If aci-GTH-A and -D contain the same hormone molecules and if the lower specific activity of the latter is due only to its contamination by an inactive component(s), one could expect the superposition of the dose-response curves for both preparations. Figure 3 shows that it is not the case. The superposition is seen only in the rising part of the curves, whereas the points indicating the percentage of oocytes with GVBD provoked by the two to three highest concentrations of aciGTH-D are situated higher than the corresponding points for aci-GTH-A. The application of sign test (Urbach, 1964) approves the highly significant (P < 0.01) difference in the position of these points. It can thus be concluded that there is a qualitative difference in the action of aci-GTH subfractions on toad oocyte maturation. Isoelectric focusing of female and male individual pituitary extracts. To study whether all aci-GTH isoforms differing by p1 values are present in a single pituitary and whether the sex-specific forms do exist, the extracts of individual pituitaries were submitted to isoelectric focusing followed by bioassay of gonadotropin. Figure 4 represents the profiles of gonadotropic activity obtained after isoelectric focusing of extracts of two females, two males, and of the mixture of hundreds of acetonized pituitaries of both sexes. It is seen that the pattern of gonadotropic activity distribution is essentially the same for all samples. The hormone molecules of three major peaks are characterized by the same p1 values. There are some differences in the appearance of the most acidic components. These differences are not linked to the sex of the pituitary donor, but they are correlated
AND
i t;
BURZAWA-GERARD
60 40 20
f 60 20 > I3
20
60 40 20
t a2
c c 0.5
1
5
10
Hbrmone
concen:raiion1°
l I
0.5
I
0
pg/ml
FIG. 3. Comparison of the action of aci-GTH subfractions A (0) and D (0) on Bufo viridis oocyte in rrifro maturation. The experiments have been carried out on the follicles of four different females (a,b,c,d). Some 40-80 (a.b) and 100-180 (c,d) follicles were used for each hormone concentration.
with the specific activity of pituitary extracts. The specitic activities of the pituitary powder from P 1, C? 1, and mixture powder were higher than that of the pituitaries from 0 2 and $ 2. Experiments carried out with extracts of other acetonized or frozen individual pituitaries (data not shown) gave the same result-no sex-linked or individual differences in the spectra of aci-GTH isoforms were observed.
POLYMORPHISM
OF
STURGEON
GONADOTROPIN.
IV
FIG. 4. Profile ofgonadotropic activity after polyacrylamide gel isoelectric focusing of male, female, and mixture of both sexes pituitary extracts in the range of pH 3.5-9.5. Extracts of 500 pg of acetonized pituitary powder were applied to gel. Gonadotropic activity was determined in 3-mm gel slices by B//.~c lsiridis oocyte in ~ilro maturation test. Some 70-90 follicles were used for each paint.
DISCUSSION
Idler et ul. (197.5) were the first to reveal the functional heterogeneity of gonadotropm purified from fish pituitaries. Two salmon (Oncorhynchus keta) gonadotropin fractions separated by DEAE-cellulose chromatography were shown to act on immature trout female and male gonads with different effectiveness. This finding allowed the authors to assume the existence of sexspecific gonadotropins in salmon pituitary. This hypothesis was further checked and reton et al. (1978). They isolated and purified gonadotropin separately from salmon (0. tsclzawytscha) female and male pituitaries and showed that the obtained hormone preparations differed by their chromatographical properties, amino acid composition, and electropho-
retical mobility. No differences in the immunological properties of the “‘fe and the “male” gonadotropins were found, The “male” gonadotropin was twice iess active than the ‘“female” one in t bow trout oocyte in vitro maturation test. Marked differences in the action of female and male pituitaries with respect to e also found in female and male gonads another teleostean fish, (Sc~~~~t~~al~~l4§ maeoticus), and two of three gonadotropin fractions revealed by disc electrophoresis were shown to be sex urlakov el ai., 1 To study whether the structuraE heterogeneity of the starred sturgeon (A. strli~lus) gonadotropin is reflected in the f~~cti~~al ur subfractions (acione we studied separated by chromaGTH-A, -B, -C, tography on BEAM-cellulose that co~ta~~e~
380
GONCHAROV,
KUZNETZOV,
differently charged hormone molecules in different proportion (Kuznetzov et al., 1983). Two of them (aci-GTH-A and -D) were practically free of common components and were studied to a greater extent in the present paper. As the studied hormone subfractions were purified from the mixture of the pituitaries of fishes of both sexes, one could surely expect to find the differences in their biological action. Surely, that is, with two “ifs” in mind-if the sexspecific forms of gonadotropins do exist in sturgeon pituitaries and if they can be separated by DEAE-cellulose chromatography. The difference was indeed found by the comparison of the specific activities of aci-GTH subfractions with different test systems. Aci-GTH-A was shown to be about twice more active than aci-GTH-D with respect to frog oocyte in vitro maturation, while both preparations manifested about equal specific activity in frog spermiation test. By this fact the idea of the existence of the sex-specific gonadotropins in fishes was further supported, but not proved. To have the direct evidence we carried out isoelectric focusing of the extracts of the individual female and male pituitaries followed by gonadotropic activity localization with toad oocyte in vitro maturation test. This method was shown to be very useful for the discrimination of aci-GTH isoforms (Goncharov et al., 1980). No sexlinked differences were found. Three major fractions were always present. The appearance in some cases of the minor fractions was due to the greater starting gonadotropic activity of the pituitary extract applied to gel rather than to the sex of the pituitary donor. The coincidence of the spectra of aciGTH isoforms in individual pituitaries also shows that the polymorphism of pituitary gonadotropin can not be due to the genetic heterogeneity of animal population. Our results do not rule out the possibility of the occurrence of the different ratio of
AND
BURZAWA-GERARD
the amount of pituitary gonadotropin isoforms related to the sex and/or to the stage of sex cycle and/or to the endocrine status of organism, as shown for mammals (Robertson et al., 1977; Wakabayashi, 1977, 1980). If it is so, the use of the low sensitive test systems that reveals only the predominant isoforms can lead to the misinterpretation of the difference in their spectra. It therefore seems premature to accept the existence of sex-specific gonadotropins in fishes. The qualitative difference in the biological action of aci-GTH-A and -D was shown in the present work by the comparison of their specific activities determined not only with different test reactions (oocyte maturation and spermiation) of the same species, but also with the same reaction (oocyte maturation) of follicles of different species. It is noteworthy that the most accentuated difference in the specific activity of aci-GTH-A and -D was revealed with the homologous follicles. It is now assumed (see Bogdanove and Nansel, 1978; Wakabayashi, 1980) that several gonadotropin isoforms can participate in the regulation of gonad function in different ways, but there is no data on the qualitative difference of their action on the target organs. To study this possibility we have chosen the amphibian follicles because of their availability and their particular properties already described under Results. Moreover, we can now speculate on the mode of gonadotropin action and try to explain the revealed difference in the action of different gonadotropins. It is well known that the stimulatory effect of gonadotropin on amphibian (Masui, 1967; Schuetz, 1967) and sturgeon (Dettlaff and Skoblina, 1969) oocyte maturation is mediated by progesterone or a related compound produced by follicular cells. At certain physiological state of gonads the inhibitory effect of gonadotropin on the progesterone-induced amphibian and sturgeon oocyte maturation can be observed (Gon-
POLYMORPHISM
OF STURGEON
1969, 1972b; Snyder, 1979). It seems very likely that the inhibitory effect of gonadotropin is mediated by CAMP that is also produced by follicular cells (Snyder, 1979; Goncharov, 1980). It can be assumed thus that at a certain stage of follicle development the quantitative ratio of these two compounds produced by follicular cells under gonadotropin influence may determine whether the oocyte undergoes maturation or not. This ratio seems to depend not only on the physiological state of follicular cells, but also on the nature of gonadotropin used. The latter can be the most plausible explanation of the fact that the higher percentage of GVBD in R. ridibunda oocytes can be obtained under the influence of sturgeon pituitary suspension as compared with the action of the homologous pituitary extract (Goncharov, 1971b). The direct evidence of the influence of the chemical structure of gonadotropin on progesterone and CAMP production was shown by Bahl (1977). The successive cleavage of carbohydrate residues of HCG changed the ratio of their production by granulosa cells. It can be suggested thus that the unknown differences in the structure of the aci-GTH isoforms causing or accompanying the charge heterogeneity of molecules also influence the ratio of progesterone and P production, which in turn determines the observed difference in the pattern of dose-response relationships obtained ubfractions (A and D) acting viridis follicles. eported in the present paper only indirectly indicate the possibility of the regulation of sturgeon gonad function by the different gonadotropin isoforms. We do not know which isoforms are secreted in the blood, reach the target organs and act on them. The qualitative difference in the action of aci-GTW subfractions was proposed to be related to the difference in the chemical structure of gonadotropin isoforms) ut special study seems to be neces-
chaTsv,
GONADOTROPIK.
IV
sary to rule out the possibility of T COl-itamination an its i~terve~ti~~ in gonadotropin action. The precautio interpretation must be taken heterologous model s ems are used for the study of pituitary rmone action (see Fontaine, 1976) ~ All now under examination.’
The authors thank 1. Dibrova for excellent technical assistance and Dr. S. G. Vassetzky for reading the English manuscript. ERE Bahl, Om P. (1977). Human chorionic gonadotropin, its receptor and mechanism of action. Fed. Proc. 36,?119-2127. Bogdanove, E. M., and Nansel, D. I). (1978:, Bioiogicai and immunological distinctions between pltuitary and serum EH in the rat. 01 ‘“Structxe and Function of the Gonadotropins” (K. W. McKerns, ed.), pp. 415-430. Plenum Press, New York; London. Breton, B., Prunet, P., and einaud, P. (19781. Sexual differences in salmon onadotropin. Afrrr. Bj~i Anim.
Bioch.
Biophys.
I$, 759-755.
Burlakov, A. B., and Gureeva-Preobrazhenskaya. E. V. (1977). Dynamics 5f the gonadotropin cortent in the hypophysis of female silver carp Hypop/~rlzalmichtl~);s molitrix (Val,) at different stages of gonad maturity. Vopr. brhtioiogii 17, 952-956 (in Russian). Burlakov, A. B., Zolotnitskii, A. P., and Moiseeva. E. B. (1979). Sexual differences of gonadotropins from the Black Sea plaice Scopi~fhc!n~~is maeoricrls.
Zh. h:o/.
Bioch.
Physiol.
65. 496-499
(in Russian). Burzawa-Gerard, E., Goncharov: Y. A. (1975). L’hormone gonadotrope hypophysaire d’un Poisson Chondrosteen, :‘Estargeon (Acipenser stellatus Pail.). I. Purification. Gen.
Comp.
Emfocrinol.
27, 289-295.
Dettlaff, T. A., and Skoblina. M. N. (1969). The roie of germinal vesicle in the process of oocyte maturation in Aura and Aripejlseridue. Atln. Embryl.
Esp.
Morphol.
Suql.
1, 133-151.
Emmens, C. W. (1948). “Principles of Biological Assay.” Chapman & Hall. London. Fontaine, M. (1976). Hormones and the control of re1 The preliminary publication of the results presented in this paper was made in the wedings 0’ 1981. ase. the Academy of Sciences of the US 243-246.
382
GONCHAROV,
production Can.
in aquaculture.
KUZNETZOV,
J. Fish.
Res.
Board
33, 922-933.
Fontaine, M., et Chauvel, M. (1961). Evaluation du pouvoir gonadotrope de l’hypophyse des Poissons Teleosteens; et en particulier du S&no salar L. B diverses &apes de son developpment et de ses migrations. C.R. Acnd. Sci. 252, 822-825. Goncharov, B. F. (1969). Inhibitory effect of hypcphysis on progesterone-induced oocyte maturation in \~ilro in frogs and sturgeon. 111 “Demonstrations Presented at the IX International Embryological Conference,” pp. 31-32. Nauka, Moscow. Goncharov. B. F. (1971a). Dependence of the duration of hormone-dependent period of follicle maturation in the common frog from concentration of pituitary suspension. A new method of testing pituitaries. Onfogenesis 2, 64-67 (in Russian). Goncharov, B. F. (1971b). “A Study of Regularities of Amphibian and Sturgeon Oocytes Transition from Growth to Maturation.” Thesis Cand. Biol. Sci.. Moscow (in Russian). Goncharov, B. F. (1972a). Assay of determination of the gonadotropic activity of the hypophysis of Acipenseridue by the reaction of oocyte maturation in vitro. In “Acipenseridae and the Problems of the Sturgeon Pisciculture” (U. U. Marti and I. A. Barannikova, eds.), pp. 257-262. Pisch. Prom., Moscow (in Russian). Goncharov, B. F. (1972b). A study of the inhibitory effect of pituitary on progesterone-induced oocyte maturation in Rarza remporaria. Gen. Comp. /37docrinol. 18, 593. Goncharov. B. F. (1980). The inhibitory effect of pituitary gonadotropic hormone on the progesteroneinduced maturation of amphibian and sturgeon oocytes. In “Poster Communications of the XII International Embryological Conference,” p. 69. Patras , Greece. Goncharov, B. F., Kuznetzov, A. A., and BurzawaGerard, E. (1980). Analysis of heterogeneity of pituitary gonadotropin from sevryuga (Acipenser
AND
BURZAWA-GERARD
Pall.). Biochemisiry (USSR) 45, 455-462 (in Russian). Idler, D. R., Bazar, I. S., and Hwang. S. 1. (19753. Fish gonadotropin(s). III. Evidence for more than one gonadotropin in chum salmon pituitary glands. Endocrine Res. Cammar;. 2, 237-249. Kuznetzov, A. A., Goncharov. B. F.. and BurzawaGerard, E. (1983). Pituitary gonadorropic hormone of a chondrostean fish, starred sturgeon (Acipenser stellatus Pall.) III. Polymorphism. stellutus
Gen.
Comp.
Endocrinoi.
49. 364-374.
Masui, U. (1967). Relative roles of the pituitary. follicle cells and progesterone in the induction of oocyte maturation in Rarta pipiens. J. Ex0. Zool. 166, 365-376. Robertson, D. M., Van Damme. M.-P., and Diszfalusy, E. (1977). Biological and irnrn~~o~~gic~ characterization of human luteinizing hormone. I. Biological profile in pituitary and pla.sma samples after electrofocusing. Mol. Celi. Endocfifiai. 9, 45-56. Schuetz, A. W. (1967). Action of hormones on germinal vesicle breakdown in frog @ana pipiew) oocytes. I, Exp. Zool. 166, 347-354. Snyder, B. W. (1979). Seasonal change in the response of Rana pipierls ovarian follicles to gonadotropic hormone. Amer. Zool. 19, 938. Thornton, V. F. (1971). A bioassay for progesterone and gonadotropins based on meiotic division of Xenopus oocytes in :,itro. Gex. Comp. Endocrinol.
16, 599-605.
Urbach. V. U. (1964). “ iometric Methods.” Nauka, Moscow (in Russian). Wakabayashi. K. (1977). Neterogeneity of rat luteinizing hormone revealed by radioimmunoassay and eiectrofocusing studies. E/~doiuinol. Japan.
24, 473-485.
Wakabayashi. K. (1980). Multiplicity of luteinizing hormone and its comparative aspects. I.0 *'Normones. Adaptation and Evolution” (S. Ishii et ni., eds.) pp. 271-279. Japan Sci. Sot. Press, Tokyo/Springer-Vet-lag. Berlin.
AND
Pituitary
COMPARATIVE
ENDOCRINOLOGY
49, 375-382 (1983)
onadotropic Hormone from Sturgeon (Acipenser s IV. Differences
B. F. GONCHAROV,
in Biological Action of Sex-specific Forms
A. A. KUZNETZOV,
AND E. BURZAWA-GERARD*
N. K. Koltrov Institute of Developmental Biology, USSR Academy of Sciences, Moscow 117 808, USSR, and *Laboratoire de Physiotogie g&&ale et comparie, d’Endocrinologie comparPe associ4 au CNRS, MusCum national d’ffistoire 7 rue Cwier, 75230 Paris Cedex 5, France
26 Vavilov St., L~boratoi,re narur-cl/u.
Accepted March 30: 1982 The specific activities of four subfractions of the starred sturgeon (Acipenser stellarus Pall.) pituitary gonadotropin (aci-GTH-A, -B, -C, -D) were compared using several test systems. The ratios of two of them (A and D) that contained the isoforms differing by the values of isoelectric point were shown to be different, especially when irz vitro oocyte maturation and spermiation tests were used. These differences were not due to the presence in one of these preparations of the component affecting spermiation rather than oocyte maturation. The qualitative differences in the biological action of aci-GTH-A and -D were also revealed by the comparison of dose-response curves using toad oocyte irr vitro maturation. Finally, as the spectra of aci-GTH isoforms were practically identical when female or male individual pituitary extracts were submitted to isoelectric focusing, we concluded that the structural and functional heterogeneity of aci-GTH was related neither to sex nor to genetic intrapopulation polymorphism.
It is now accepted that the polymorphism of vertebrate pituitary gonadotropic hormones may have a functional significance (see Bogdanove and Nansel, 1978; Wakabayashi, 1980). Interesting aspects of this problem have arisen when fish gonadotropins have been investigated. The existence of sex-specific forms of pituitary gonadotropic hormone has been suggested (Idler et ai., 1975; Breton et ai., 1978; Burlakov et ai., 1979). An attempt has also been made to elucidate the relationship between the relative amount of electrophoretically different gonadotropic fractions in the carp pituitary and the stage of ovarian development (Burlakov and Gureeva-Preobrazhenskaya, 1977). In the accompanying paper (Kuznetzov et al., 1983) we described the purification of four active subfractions of sturgeon pituitary gonadotropin (a>H) that differed by the relative content of differently charged
isoforms. The present work was done to study whether these differently isoforms were identical by their b properties. Two kinds of approaches were used to answer this question. First was the comparison of the ratio of the specific activities of the studied hormone preparations determined with different test systems. As the second approach, we used the comparison of the form of dose-response curves using amphibian oocyte in ~12ro rna~~rat~o~. In both cases the qualitative differences in the biological action of two aci-GTH subfractions extreme by isoelectric point (p1) values of their molecules were revealed. We also compared the GTH isoforms in the indiv of female and male fishes significant differences. MATERIALS
AND METHO
Four subfractions of aci-GTE (A,B.C.D; were studied in the present work. Their purification. Materials.
375 0016.6480/83/Q30375-08$0i.5o:o Copyright All rights
@ 1983 by Academic Press. inc. of reproduction in any form resewed
376
GONCHAROV,
KUZNETZOV,
separation, and some physical and chemical properties are described elsewhere (Kuznetzov et al., 1983). Individual pituitary glands were collected from sexually mature female and male sturgeons (Acipenser stellatlds Pall.) caught in the Volga river during their anadromous migration. After dissection pituitaries were either acetonized or frozen and kept at -20 until use. Bioassa~s. Amphibian and sturgeon oocyte in vitro maturation test (Thornton, 1971; Goncharov, 1971a, 1972a) was used as earlier described in detail (BurzawaGerard et al., 1975). Frog (Rarra tempornuia) spermiation test was realized in the conditions described elsewhere (Fontaine and Chauvel, 1961). In addition, two methods of registration of spermiation were used: qualitativewhen the presence or the absence of spermiation was registered, and semiquantitative-when the intensity of reaction was estimated and expressed in arbitrary units (1, weak reaction-single spermatozoa are in the sample: 2, middle reaction-not more than 10 spermatozoa are seen in the field of vision of microscope at x 100 magnification: 3, strong reaction-more than 10 spermatozoa are seen under the same conditions). Isoelectric focusing. Analytic polyacrylamide gel isoelectric focusing of the extracts of individual pituitaries followed by the determination of gonadotropic activity distribution using the toad (Bufo viridis) oocyte in vitro maturation test was carried out as de-
I
OOCYTE Acipenser steilotus
AND BURZAWA-GERARD scribed earlier (Kuznetzov et al., 1983). When the frog spermiation test was used to localize the gonadotropic activity after isoelectric focusing, gel was sliced in 3-mm fragments. Each fragment was then placed in a flacon containing 2.5 ml of amphibian Ringer solution. After the overnight elution at 4”, the OS-ml aliquots were injected into the male frogs. Stutistical methods. The median effective dose (ED,,) and/or potency ratios were calculated with limits for P = 0.05 according to Emmens (1948). Sign test was used according to Urbach (1964).
RESULTS
Comparison of specific activities of four aci-GTH subfractions using several test systems. In Fig. 1 are shown the relative potencies of four aci-GTH subfractions (A,B,C,D) as compared by their ED,,. According to in vitro oocyte maturation test (whatever species was used), three aciGTH preparations (A,B,C) were of about equal specific activity, while aci-GTH-D was 2-3 times less active. On the contrary, the frog spermiation test did not reveal any significant differences between all aciGTH subfractions whatever method of reg-
s PERMIATION
MATURATION Bufo vi ridis
RQIICI temporario
[email protected]
I
i 20
temporaria
FIG. 1. Comparison of specific activities of four subfractions of aci-GTH (A,B,C,D) determined by several bioassays. In the case of irz vitro oocyte maturation test the experiments have been repeated with follicles of two females of each species. 1, qualitative, and 2, semiquantitative method of registration of spermiation reaction (see Materials and Methods for details). Limits for P = 0.05.
POLYMORPHISM
SPECIFK
ACTIVITY
RATIO
Test reaction:
Species: 9 1 92 Note.
Calculations
OF
STURGEON
GONADOTROPIN.
TABLE aci-GTH-Naci-GTH-D In vitro
oocyte
1 DETERMINED
BY SEYER+L
Bro~ss~ys
maturation
Spermiation
Acipemer stellarus
Rar1a
ternporaria
3.1 (2.6-3.8) 2.9 (2.5-3.4) are done from
34?
IV
1.7 (1.5-2.0) 1.9 (1.7-2.2) the results
presented
1.7 (1.5-1.9) 2.1 (1.8-2.5) irr Fig.
1. I+, ?+ please
‘+l.l (W-3.4) Z-1.1 (0.9-1.3) see legend
to Fig.
i. Liinits
cob;
P = Q.05.
istration-qualitative or semiquantitative (see Materials and Methods) was used. To avoid the inaccuracy, the same preliminary frozen stock aci-GTH preparations solutions were used for the comparison of then- specific activities with R. temporaria oocyte maturation and spermiation tests. Thus, the significant discrepancy in the relative potency of aci-GTH-A and -D was with different test-systems used (Table I). Cornparisotz of the profiles of gonadotropic activity of aci-GTH-D submitted to isoelectric .focusing as determined by oocyte maturation and sperrniation test. The
observed discrepancy in the ratios of specific activities of aci-GTH-A and -D obtamed with oocyte maturation and spermiation tests could be explained by the fact that aci-GTHcontained a component which manifested itself only or predominantly in the spermiation test. However, when the eluates of gel fragments obtained after isoelectric focusing of aci-GTH-D were tested with toad oocyte in vitro maturation or frog spermiation, no component predominantly acting on spermiation was observed (Fig. 2). On the contrary, the isoforms of aci-GTH with higher pZ values were revealed by oocyte maturation rather than by spermiation test. But it seems to be due to the low-er sensitivity of the frog spermiation test (see Fig. 1) rather than to the characteristics of these components. Comparison of dose-response curves for aci-GTH-.4 and -D. This experiment was
carried out to try to reveal qualitative
dif-
ferences in the action of acion toad oocyte maturation. us (Goncharov, 1971b) sh ~~ysio~ogica~ state of large follicles varied markedly in the hibernat late-spawning amphibians. cles of some of them were exposed ii? 13r.o to the increasing co~ce~t~at~o~ of pituitary suspension, the percentage of oocytes with GVBD (germinal vesicle breakdowns raised, reached the maximal level ~d~ffer~~t fior different females) and then went The maximal percentage of matur cytes that can be obtained as w of the dose-response curve v spect to species from which taken. This model system was used to corn the dose-response relationships of two 100
50
0
75
SC
5.5
50
p:’
2. Profile of gonadotropic activity after polyacrylamide gel isoeiectric focusing of aci-GTil-D in the range of pH 3.5-9.5. Gonadotropic activity determined by two bioassays: 1 (0) Bufo viridi$ oocyte 1~ Iaitro maturation (50 pg of protein were applied to gel: 40-60 follicles were used for each point): and 2 ( Rana temporaria spermiation (250 pg of protein were applied to gel: 5 males were used for each point). Ordinate-percentage cf oocytes with GVB.D (germinal vesicle breakdown) or frog males responded by spermiation. FIG.
378
GONCHAROV,
KUZNETZOV,
aci-GTH subfractions (A and D). The follicles of four toad females were used for this kind of experiment. The results are presented in Fig. 3. As the specific activity of aci-GTH-A is approximately twice higher than that of aci-GTH-D, the dose scales are shifted correspondingly. If aci-GTH-A and -D contain the same hormone molecules and if the lower specific activity of the latter is due only to its contamination by an inactive component(s), one could expect the superposition of the dose-response curves for both preparations. Figure 3 shows that it is not the case. The superposition is seen only in the rising part of the curves, whereas the points indicating the percentage of oocytes with GVBD provoked by the two to three highest concentrations of aciGTH-D are situated higher than the corresponding points for aci-GTH-A. The application of sign test (Urbach, 1964) approves the highly significant (P < 0.01) difference in the position of these points. It can thus be concluded that there is a qualitative difference in the action of aci-GTH subfractions on toad oocyte maturation. Isoelectric focusing of female and male individual pituitary extracts. To study whether all aci-GTH isoforms differing by p1 values are present in a single pituitary and whether the sex-specific forms do exist, the extracts of individual pituitaries were submitted to isoelectric focusing followed by bioassay of gonadotropin. Figure 4 represents the profiles of gonadotropic activity obtained after isoelectric focusing of extracts of two females, two males, and of the mixture of hundreds of acetonized pituitaries of both sexes. It is seen that the pattern of gonadotropic activity distribution is essentially the same for all samples. The hormone molecules of three major peaks are characterized by the same p1 values. There are some differences in the appearance of the most acidic components. These differences are not linked to the sex of the pituitary donor, but they are correlated
AND
i t;
BURZAWA-GERARD
60 40 20
f 60 20 > I3
20
60 40 20
t a2
c c 0.5
1
5
10
Hbrmone
concen:raiion1°
l I
0.5
I
0
pg/ml
FIG. 3. Comparison of the action of aci-GTH subfractions A (0) and D (0) on Bufo viridis oocyte in rrifro maturation. The experiments have been carried out on the follicles of four different females (a,b,c,d). Some 40-80 (a.b) and 100-180 (c,d) follicles were used for each hormone concentration.
with the specific activity of pituitary extracts. The specitic activities of the pituitary powder from P 1, C? 1, and mixture powder were higher than that of the pituitaries from 0 2 and $ 2. Experiments carried out with extracts of other acetonized or frozen individual pituitaries (data not shown) gave the same result-no sex-linked or individual differences in the spectra of aci-GTH isoforms were observed.
POLYMORPHISM
OF
STURGEON
GONADOTROPIN.
IV
FIG. 4. Profile ofgonadotropic activity after polyacrylamide gel isoelectric focusing of male, female, and mixture of both sexes pituitary extracts in the range of pH 3.5-9.5. Extracts of 500 pg of acetonized pituitary powder were applied to gel. Gonadotropic activity was determined in 3-mm gel slices by B//.~c lsiridis oocyte in ~ilro maturation test. Some 70-90 follicles were used for each paint.
DISCUSSION
Idler et ul. (197.5) were the first to reveal the functional heterogeneity of gonadotropm purified from fish pituitaries. Two salmon (Oncorhynchus keta) gonadotropin fractions separated by DEAE-cellulose chromatography were shown to act on immature trout female and male gonads with different effectiveness. This finding allowed the authors to assume the existence of sexspecific gonadotropins in salmon pituitary. This hypothesis was further checked and reton et al. (1978). They isolated and purified gonadotropin separately from salmon (0. tsclzawytscha) female and male pituitaries and showed that the obtained hormone preparations differed by their chromatographical properties, amino acid composition, and electropho-
retical mobility. No differences in the immunological properties of the “‘fe and the “male” gonadotropins were found, The “male” gonadotropin was twice iess active than the ‘“female” one in t bow trout oocyte in vitro maturation test. Marked differences in the action of female and male pituitaries with respect to e also found in female and male gonads another teleostean fish, (Sc~~~~t~~al~~l4§ maeoticus), and two of three gonadotropin fractions revealed by disc electrophoresis were shown to be sex urlakov el ai., 1 To study whether the structuraE heterogeneity of the starred sturgeon (A. strli~lus) gonadotropin is reflected in the f~~cti~~al ur subfractions (acione we studied separated by chromaGTH-A, -B, -C, tography on BEAM-cellulose that co~ta~~e~
380
GONCHAROV,
KUZNETZOV,
differently charged hormone molecules in different proportion (Kuznetzov et al., 1983). Two of them (aci-GTH-A and -D) were practically free of common components and were studied to a greater extent in the present paper. As the studied hormone subfractions were purified from the mixture of the pituitaries of fishes of both sexes, one could surely expect to find the differences in their biological action. Surely, that is, with two “ifs” in mind-if the sexspecific forms of gonadotropins do exist in sturgeon pituitaries and if they can be separated by DEAE-cellulose chromatography. The difference was indeed found by the comparison of the specific activities of aci-GTH subfractions with different test systems. Aci-GTH-A was shown to be about twice more active than aci-GTH-D with respect to frog oocyte in vitro maturation, while both preparations manifested about equal specific activity in frog spermiation test. By this fact the idea of the existence of the sex-specific gonadotropins in fishes was further supported, but not proved. To have the direct evidence we carried out isoelectric focusing of the extracts of the individual female and male pituitaries followed by gonadotropic activity localization with toad oocyte in vitro maturation test. This method was shown to be very useful for the discrimination of aci-GTH isoforms (Goncharov et al., 1980). No sexlinked differences were found. Three major fractions were always present. The appearance in some cases of the minor fractions was due to the greater starting gonadotropic activity of the pituitary extract applied to gel rather than to the sex of the pituitary donor. The coincidence of the spectra of aciGTH isoforms in individual pituitaries also shows that the polymorphism of pituitary gonadotropin can not be due to the genetic heterogeneity of animal population. Our results do not rule out the possibility of the occurrence of the different ratio of
AND
BURZAWA-GERARD
the amount of pituitary gonadotropin isoforms related to the sex and/or to the stage of sex cycle and/or to the endocrine status of organism, as shown for mammals (Robertson et al., 1977; Wakabayashi, 1977, 1980). If it is so, the use of the low sensitive test systems that reveals only the predominant isoforms can lead to the misinterpretation of the difference in their spectra. It therefore seems premature to accept the existence of sex-specific gonadotropins in fishes. The qualitative difference in the biological action of aci-GTH-A and -D was shown in the present work by the comparison of their specific activities determined not only with different test reactions (oocyte maturation and spermiation) of the same species, but also with the same reaction (oocyte maturation) of follicles of different species. It is noteworthy that the most accentuated difference in the specific activity of aci-GTH-A and -D was revealed with the homologous follicles. It is now assumed (see Bogdanove and Nansel, 1978; Wakabayashi, 1980) that several gonadotropin isoforms can participate in the regulation of gonad function in different ways, but there is no data on the qualitative difference of their action on the target organs. To study this possibility we have chosen the amphibian follicles because of their availability and their particular properties already described under Results. Moreover, we can now speculate on the mode of gonadotropin action and try to explain the revealed difference in the action of different gonadotropins. It is well known that the stimulatory effect of gonadotropin on amphibian (Masui, 1967; Schuetz, 1967) and sturgeon (Dettlaff and Skoblina, 1969) oocyte maturation is mediated by progesterone or a related compound produced by follicular cells. At certain physiological state of gonads the inhibitory effect of gonadotropin on the progesterone-induced amphibian and sturgeon oocyte maturation can be observed (Gon-
POLYMORPHISM
OF STURGEON
1969, 1972b; Snyder, 1979). It seems very likely that the inhibitory effect of gonadotropin is mediated by CAMP that is also produced by follicular cells (Snyder, 1979; Goncharov, 1980). It can be assumed thus that at a certain stage of follicle development the quantitative ratio of these two compounds produced by follicular cells under gonadotropin influence may determine whether the oocyte undergoes maturation or not. This ratio seems to depend not only on the physiological state of follicular cells, but also on the nature of gonadotropin used. The latter can be the most plausible explanation of the fact that the higher percentage of GVBD in R. ridibunda oocytes can be obtained under the influence of sturgeon pituitary suspension as compared with the action of the homologous pituitary extract (Goncharov, 1971b). The direct evidence of the influence of the chemical structure of gonadotropin on progesterone and CAMP production was shown by Bahl (1977). The successive cleavage of carbohydrate residues of HCG changed the ratio of their production by granulosa cells. It can be suggested thus that the unknown differences in the structure of the aci-GTH isoforms causing or accompanying the charge heterogeneity of molecules also influence the ratio of progesterone and P production, which in turn determines the observed difference in the pattern of dose-response relationships obtained ubfractions (A and D) acting viridis follicles. eported in the present paper only indirectly indicate the possibility of the regulation of sturgeon gonad function by the different gonadotropin isoforms. We do not know which isoforms are secreted in the blood, reach the target organs and act on them. The qualitative difference in the action of aci-GTW subfractions was proposed to be related to the difference in the chemical structure of gonadotropin isoforms) ut special study seems to be neces-
chaTsv,
GONADOTROPIK.
IV
sary to rule out the possibility of T COl-itamination an its i~terve~ti~~ in gonadotropin action. The precautio interpretation must be taken heterologous model s ems are used for the study of pituitary rmone action (see Fontaine, 1976) ~ All now under examination.’
The authors thank 1. Dibrova for excellent technical assistance and Dr. S. G. Vassetzky for reading the English manuscript. ERE Bahl, Om P. (1977). Human chorionic gonadotropin, its receptor and mechanism of action. Fed. Proc. 36,?119-2127. Bogdanove, E. M., and Nansel, D. I). (1978:, Bioiogicai and immunological distinctions between pltuitary and serum EH in the rat. 01 ‘“Structxe and Function of the Gonadotropins” (K. W. McKerns, ed.), pp. 415-430. Plenum Press, New York; London. Breton, B., Prunet, P., and einaud, P. (19781. Sexual differences in salmon onadotropin. Afrrr. Bj~i Anim.
Bioch.
Biophys.
I$, 759-755.
Burlakov, A. B., and Gureeva-Preobrazhenskaya. E. V. (1977). Dynamics 5f the gonadotropin cortent in the hypophysis of female silver carp Hypop/~rlzalmichtl~);s molitrix (Val,) at different stages of gonad maturity. Vopr. brhtioiogii 17, 952-956 (in Russian). Burlakov, A. B., Zolotnitskii, A. P., and Moiseeva. E. B. (1979). Sexual differences of gonadotropins from the Black Sea plaice Scopi~fhc!n~~is maeoricrls.
Zh. h:o/.
Bioch.
Physiol.
65. 496-499
(in Russian). Burzawa-Gerard, E., Goncharov: Y. A. (1975). L’hormone gonadotrope hypophysaire d’un Poisson Chondrosteen, :‘Estargeon (Acipenser stellatus Pail.). I. Purification. Gen.
Comp.
Emfocrinol.
27, 289-295.
Dettlaff, T. A., and Skoblina. M. N. (1969). The roie of germinal vesicle in the process of oocyte maturation in Aura and Aripejlseridue. Atln. Embryl.
Esp.
Morphol.
Suql.
1, 133-151.
Emmens, C. W. (1948). “Principles of Biological Assay.” Chapman & Hall. London. Fontaine, M. (1976). Hormones and the control of re1 The preliminary publication of the results presented in this paper was made in the wedings 0’ 1981. ase. the Academy of Sciences of the US 243-246.
382
GONCHAROV,
production Can.
in aquaculture.
KUZNETZOV,
J. Fish.
Res.
Board
33, 922-933.
Fontaine, M., et Chauvel, M. (1961). Evaluation du pouvoir gonadotrope de l’hypophyse des Poissons Teleosteens; et en particulier du S&no salar L. B diverses &apes de son developpment et de ses migrations. C.R. Acnd. Sci. 252, 822-825. Goncharov, B. F. (1969). Inhibitory effect of hypcphysis on progesterone-induced oocyte maturation in \~ilro in frogs and sturgeon. 111 “Demonstrations Presented at the IX International Embryological Conference,” pp. 31-32. Nauka, Moscow. Goncharov. B. F. (1971a). Dependence of the duration of hormone-dependent period of follicle maturation in the common frog from concentration of pituitary suspension. A new method of testing pituitaries. Onfogenesis 2, 64-67 (in Russian). Goncharov, B. F. (1971b). “A Study of Regularities of Amphibian and Sturgeon Oocytes Transition from Growth to Maturation.” Thesis Cand. Biol. Sci.. Moscow (in Russian). Goncharov, B. F. (1972a). Assay of determination of the gonadotropic activity of the hypophysis of Acipenseridue by the reaction of oocyte maturation in vitro. In “Acipenseridae and the Problems of the Sturgeon Pisciculture” (U. U. Marti and I. A. Barannikova, eds.), pp. 257-262. Pisch. Prom., Moscow (in Russian). Goncharov, B. F. (1972b). A study of the inhibitory effect of pituitary on progesterone-induced oocyte maturation in Rarza remporaria. Gen. Comp. /37docrinol. 18, 593. Goncharov. B. F. (1980). The inhibitory effect of pituitary gonadotropic hormone on the progesteroneinduced maturation of amphibian and sturgeon oocytes. In “Poster Communications of the XII International Embryological Conference,” p. 69. Patras , Greece. Goncharov, B. F., Kuznetzov, A. A., and BurzawaGerard, E. (1980). Analysis of heterogeneity of pituitary gonadotropin from sevryuga (Acipenser
AND
BURZAWA-GERARD
Pall.). Biochemisiry (USSR) 45, 455-462 (in Russian). Idler, D. R., Bazar, I. S., and Hwang. S. 1. (19753. Fish gonadotropin(s). III. Evidence for more than one gonadotropin in chum salmon pituitary glands. Endocrine Res. Cammar;. 2, 237-249. Kuznetzov, A. A., Goncharov. B. F.. and BurzawaGerard, E. (1983). Pituitary gonadorropic hormone of a chondrostean fish, starred sturgeon (Acipenser stellatus Pall.) III. Polymorphism. stellutus
Gen.
Comp.
Endocrinoi.
49. 364-374.
Masui, U. (1967). Relative roles of the pituitary. follicle cells and progesterone in the induction of oocyte maturation in Rarta pipiens. J. Ex0. Zool. 166, 365-376. Robertson, D. M., Van Damme. M.-P., and Diszfalusy, E. (1977). Biological and irnrn~~o~~gic~ characterization of human luteinizing hormone. I. Biological profile in pituitary and pla.sma samples after electrofocusing. Mol. Celi. Endocfifiai. 9, 45-56. Schuetz, A. W. (1967). Action of hormones on germinal vesicle breakdown in frog @ana pipiew) oocytes. I, Exp. Zool. 166, 347-354. Snyder, B. W. (1979). Seasonal change in the response of Rana pipierls ovarian follicles to gonadotropic hormone. Amer. Zool. 19, 938. Thornton, V. F. (1971). A bioassay for progesterone and gonadotropins based on meiotic division of Xenopus oocytes in :,itro. Gex. Comp. Endocrinol.
16, 599-605.
Urbach. V. U. (1964). “ iometric Methods.” Nauka, Moscow (in Russian). Wakabayashi. K. (1977). Neterogeneity of rat luteinizing hormone revealed by radioimmunoassay and eiectrofocusing studies. E/~doiuinol. Japan.
24, 473-485.
Wakabayashi. K. (1980). Multiplicity of luteinizing hormone and its comparative aspects. I.0 *'Normones. Adaptation and Evolution” (S. Ishii et ni., eds.) pp. 271-279. Japan Sci. Sot. Press, Tokyo/Springer-Vet-lag. Berlin.