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The gonadotropins of lower vertebrates
GlNERAL
AND
COMPARATIVE
ENDOCRINOLOGY
SUPPLEMENT
The Gonadotropins
de Physiologie laboratoire
715-728
of lower
E. BURZAWA-GERARD Laboratoire nature&;
3,
AND
(197%
Vertebrates1
Y. A. FONTAINE
ge’ne’rale et compare’e du Mu&urn d%ndocrinologie compare’e associe’ Y, rue Cuvier, Paris 5e, France
national d’llistoire au C.NR.S.,
After reviewing data on comparative biochemical and biological properties of gonadotropins (GTH) from lower vertebrates, we discuss two main problems: the number of GTHs in each lower vertebrate, and the specificity and evolutioa of GTHs. It is concluded that: (1) The existence of a single GTN in the teleost, and possibly also in the amphibian and reptile, is the most probable hypothesis. (2) While all GTHs (and TSHs) are probably homologous proteins, an important zoological specificity of GTH does exist. Enally, preliminary results suggest that the gonadal receptors for GTH in iowcr vertebrates are, as in mammals, associated with an adenyl cyclase activi;ily. The evolution of these “analogous” receptors should be considered together with that of the gonadotropins. It is suggested that the differentiation of the two classes of receptors (respectively, in gametogenetic and steroid-producing tissues) allowed a precise pituitary regulation of the gonads before the appearance of a second pituitary gonadotropin.
In all lower vertebrates, as in mammals, the gonads are under the dependence of the pituitary, even if this dependence can be less rigid in some groups, Cyclostomes for instance (see Pickford and Ate, 1957; Dodd, 1960; Fontaine, 1968; Licht, and Pearson, 1969a). This control is exerted, at least principally, by hormones which act directly upon gonadal tissue, and which are synthesized and secreted by specialized cells of the anterior pituitary. The present paper will deal with these gonadotropin hormones (GTH) but it will not be an exhaustive review due to the very large amount of information avail-
able. For instance? the eventual interference of other hormones (~~prola~t~~~‘~ steroids, etc.) in the action of be left aside. We shall main two problems: number of GT lower vertebrate, and specificity and evolution, of GTHs. In order to discuss these points we shall first describe some recent results concerning the p~r~fi~at~~~ of lower vertebrate GTHs, and the comparison of biochemical and b~~l~~i~al properties of GTHR from various vertebrates.
’ GTH : gonadotropic hormone ; LH : luteinizing hormone; FSH: follicle-stimulating hormone ; TSH: thyrotropic hormone ; HCG : human ehorionic gonadotropin; PMSG : pregnant mare serum gonadotropin. A letter, or letters preceding the name of a hormone indicates the zoological origin by referring to the latin genus name of the species considered: e.g., c = Cypri32215, o = ovine, one = Oncorhynchus; DEAE-C = diethylaminoethyl cellulose ; AMP = adenosine monophosphate. KD = exclusion coefficient on Sephadex.
So far only teleost G purified. Because, as we shall see, these hormones do not have t,ypical actions in mammals, bioassays have to be made on lower vertebrates.
I-PURIFICATION FROM
by
Academic
Press,
Inc.
GONADOTROPINS, VERTEBRATES
A. Purification of c-GTlrl This purification has been followed by measuring the gonadotropic activity frog spermiation in the conditions scribed by Fontaine and Chauvel (1961). 715
@ 1972
OF LOWER
716
BURZAWA-GERARD
AND
The first steps of the purification, which have been described in detail elsewhere (Fontaine and G&ard, 1963) are summarized in Fig. 1. The recent addition of a preparative electrophoresis on polyacrylamide gel led to a better purification (Buraawa-GBrard, 1971). Figure 2 shows that the biologically active peak is eluted with an Rf of about 0.50, and separated from an inactive material (Rf = 0.80). After recovery on Sephadex G 100, the lyophilised powder is about 20 times more active than the starting material. Its activity is 1.5 X NIH LH Sl (determination made in October). The yield in activity is about 40%. Another attempt to prepare c-GTH has been made by Clemens et al. (1964). They followed the’activity by measuring gonadal hydration in goldfish (Clemens and Grant, 1964), and used only a Sephadex G 75
FONTAINE
filtration. In spite of some apparent discrepancy it seems probable that the material they obtained corresponds to t;he same c-GTH we purified (BurzawaGlBrard and -Fontaine, 1966). B. Purification of one-GTH Donaldson and Yamazaki (1968) have purified a GTH from pituitaries of Pacific Salmon (Oncorhynchus tshawytscha) . They followed the activity by studying the induction of spermiation in hypophysectomised goldfish (Yamazaki and Donaldson, 1968a). The biochemical procedure does not seem to have been published in detail; these authors used an alcoholic extraction followed by two filtrations on Sephadex G 100, and eventually DEAE-C chromatography. The partly purified oncGTH (after G 100) had an activity of 3500
Extraction‘by AlcoholicPercolation I
1
Fractionsolohle in ( Et OH57% NaCl 2 %)
Unretardedfraction
1fi~:!wf~.“:‘“‘- C1 Adsorhedfraction eluted by gradient of ionic strenghth
Fraction of KD = 0.28 Preparative electrophoresis Fraction of RF
=
0.50 -0.54
Fractionof KD= 0.28 Lyophylisation---+x4-1 FIG. 1. Main
steps
in the purification
of c-GTH.
GONADOTROPINS
OF
717
VERTEBRATES
t-5~o~o~icalactivity in termsof NH- LHI- SIT
0
50
100
150
Effluent(ml)
FIG. 2. Preparative electrophoresis of partly purified c-GTH (after Sephsdex G 100 and I?EAE-C) on polyacrylamide gel. O-O Proteins (o.d. 276 mp). 0-0 Biological activity, measured cr, frog spermislion test, in terms of NIH-LH-Sll (pg/mt). ? -No animal responds to the inje&ed dose. 8 --All animals -respond to the injected dose. During the electrophoresis the material is concentrated in the upper gel (2.57; acrylamide), and the individual components are separated in Tris-Glycine buffer (Tris 0.36 &f, Giycine 0.043 ilf ; pH 10.3) in the resolving gel (7yc acrylamide). The migration occurs from the anode to the cathode and the proteins are &ted in Tris-WC1 bufYer pH 8.1 (0.1 Ai Tris). I .= 40 mA, V = 200 V, and el!llion rate 10 ml/hour.
IU HCG/mg (HCG being active in the bioassay can be used as a standard). II-BIOCHEMICAL PROPERTIES OF WITH MAMMALIAN
AND IMMUNOLOGICAL c-GTH. COMPARISON GONADOTROPINS
The behaviour of the biological activity on ion exchangers, and the electrophoretical properties of purified e-GTH show that it is rather an acidic protein like mammalian FSHs. The molecular weight of c-GTH has been shown by sucrose gradient centrifugation to be around 27,000-31,000 (Burzawa-Gitrard and Fontaine, 1966 ; Burzawa-C&ard, 1971) very close to that of mammalian gonadotropins (see Geschwind, 1969; Jut&z and De la Llosa, 1969). The homogeneity of purified c-GTH has
been studied by ana,lytical and preparative electrophoresis. If, for instance, the c-GTH obtained by lyophilisation of the aclive peak (RP = 0.50) in preparative electrophoresis is subjected to anot,her preparative electrophoresis, again two proteie peaks are observed, one active (Rf = O.SO), and one inactive (Idl = 0.80). The maleeular weight of this last materiaE is about half of that. of the active protein (Burzawa-G&ard? 1971). This result sugges-& that c-GTH might, be dissociable in t,wo subunits as it has been shown for mammalian LHs (see Jutisz and De la Llqsa, 1969; Geschwind, 1969), TSHs (Pierce et aE., 19X), and finally FSHs @q&off and Ekblad, 1970). Amino acid composition of c-GTH has been determined and Table I
718
BURZAWA-GERARD
COMPOSITION
OF c-GTH
AND
AND
FONTAINE
TABLE 1 COMPARISON WITH THAT
comparison with the compositions of ovine LH and FSH. It is clear that c-GTH is quite different from both mammalian FSHs and LHs. Finally, c-GTH appears to be a glycoprotein like mammalian GTHs (Table 1). c-GTH is antigenic in guinea pigs, or rabbits. Antiserum obtained from rabbits immunised against c-GTH gives in immunoelectrophoresis two precipitation arcs which probably correspond to the proteins having Rf of, respectively, 0.50 and 0.80 in acrylamide electrophoresis. On the contrary there is no precipitation between the same antiserum and o-LH or o-FSH. By use of antibodies obtained in guinea pigs, and of a labeled 13?I c-GTH, a radioimmunoassay of c-GTH has recently been established. In this system there appears to be some cross reaction bet,ween C-GTH and o-LH (Breton et .aZ.j 1971).
OF MAMMALIAN
FSH
AND
LHs
III-COMPARISON OF THE BIOLOGICAL PROPERTIES OF VARIOUS GONADOTROPINS
A. On Teleost Fish c-GTH is actually secreted in the blood as shown by radioimmunoassay (Breton et al., unpublished data). It stimulates many parameters of the gonadal function in teleost fish: hydration of the testis,2 32P uptake by eel testis (Buwawa-Gerard and Fontaine, 1965), adenyl cyclase activity in goldfish at the beginning of the vitellogenesis (Fontaine et al., 1970). By these various quantitative tests, the activity ratio c-GTH/carp pituitary is statisaWhen testing electrophoresis by activity is in frog spermiation
the effluent of a preparative this method, the maximum the same tubes as by use of the test.
GONADOTROPINS
OF
tically the same as determined in amphibians (Table 2). In hypophysectomieed goldfish, e-GTH (1 pg/g) restores spermatogenesis and spermiation in males (Billard et al., 19?0), and vitellogenesis in females (Breton et al., unpublished dat’a) . These last results agree very well with those obtained with purified one-GTH by Yamazaki and Donaldson (1968b) who, moreover, showed an action on the ovulation, and a stimulation of the act.ivity of the 3 P-ol-dehydrogenase (1969). Liley and Donaldson (1969) observed with oncGTH, stimulation of growth and maturation of the ovary in the guppy (Poecilia reticulata), and their results also suggest a stimulation of the production of an ovarian hormone involved in the regulation of sexual behavior. Menon and Smith (1971) observed an increase cyclic AMP accumulation in slices of testis from chinook salmon under the influence of one-GTH. Studies of the effects on fish of gonadotropic preparations from pituitaries of RATIO
Species
Reference
BurzawaGerard and Fontaine1965
C-GTHJCARP
FISH Anguilla anguilla Carassiusauratus
AMPHIBIANS BurzawaGirard andFontaine$965 Gurzawa Gerard1971 Ranaesculenta Hohsonand Barr1966 _ idem_ BurzawaGerard andFontaine1985
PITUITARY
2 MEASURED
Test
ON VARIOUS
RECIPIEXTS
Second preparatiea ( BurzawaGerard 1971)
First preparation (FontaineandGsrard 1963)
11.6 *
32P uptakebythe testis (4.4
Fontaineandal.rno
n.9
amphibian and reptiles gave conflicting results. The same is true with mammalia,n LH, while mammalian FSH is generally inactive (see Pickford and Atz, 1957; Hoar, 1966; Sundararaj and Nayyar, 1967; Goswami and Sundararaj, 1968). The stimulation of various aspects of gonadxi activity by LH has been observed for instance by Ashen and Hoar (1963)) Yamazaki (1965), Wiebe (1969)) but LH has no action on spermiation (Yamazaki and Donaldson, 1968a) ) or on spermatogenesis (Billard et al., 1970) in the hypophysectomized goldfish. In quantitative bioassays (hydration of the testis, 32P testis uptake) mammalian gonadotropins have either no action, or a low non-typical action (which does not increase with the dose) (Clemens and Johnson, 1964; Burzawa-G,Brard and Fontaine, 1965 ; Burzawa-Gerard, 1969) _ No action of mammalian LH and FSH is seen on goldfish ovarian adenyl cyclase either in quiescent ovaries or at t.he end of the spa-wning, while c-GTE is active in both cases (Fontaine et al., 197%; I?ontaine et ai., unpublished data). Positive
TABLE ACTIVITY
VERTEBRATES
- 30.3)
20
ovarianadenylcyclase
Spermiation
6.8 (4.3
5ana pipiens*++
Spermiation
6.6
Xenopuslaevis’*
Spermiation
11.2 7.4”
/ Alytesobstetricans Swellingof the testis (0.23
15 *:
*
- 10.7)
(Ia
1
- 41.7) I
* Limits for p= 0.95
=**Kindly testedby 5 Barr
- 22 )
720
BURZAWA-GERARD
results have been more consistently obtained with human chorionic gonadotropin. HCG determines the maturation of gonads in male eels (Fontaine, 1936) but not in female eels where c-GTH does so (Fontaine et al., 1964; Burzawa-Gerard, 1969). In other species of teleosts HCG is active on various aspects of the gonadal function, both in males (Sundararaj and Nayyar, 1967; Yamazaki and Donaldson, 1968a; Hyder et al., 1970)) and in females (Yamazaki, 1965; Goswami and Sundararaj, 1968). Mammalian gonadotropins might stimulate some aspects of gonadal function not directly but by the intermediary of a heterotropic action. It is known that mammalian gonadotropins can stimulate teleost thyroids (Fontaine, 1969a). Similarly, papers from Sundararaj and Goswami (1966a and b, 1969)) and Nayyar and Sundararaj (1969) show that LH acts upon the secretory activity of the interrenal in the catfish; in turn corticosteroids would be responsible for the observed action of LH on ovulation and spawning, as previously shown (see Ramaswami, 1962). B. On Amphibians,
Reptiles, and
Mammals
As far as the amphibians are concerned we shall limit ourselves to the studies done on males. Extracts of teleost pituitaries can induce spermiation in the frogs Rana esculenta (Fontaine and Chauvel, 1961), and Xenopus Zaewis (Barr and Hobson, 1964). During the fractionation of carp pituitary by various biochemical methods the activity was followed by the frog spermiation test. In any case only one active peak was found. Our results suggest that in carp pituitary only the carp gonadotropin c-GTH is active on frog spermiation at least in the conditions described by Fontaine and Chauvel (1961). Kihlstrom and Danninge (1970) have suggested that this response can be elicited by factors present in frog pituitary which are not the gonadotropins on the basis of the positive results they obtained after
AND
FONTAINE
extraction of frog pituitaries at 100” by acetic acid 0.25%, and evaporation. Indeed we have obtained somewhat similar results with carp pituitary; however quantitatively only 10% of the original activity is present after this treatment; the nature of this active material has to be investigated further but it does not appear impossible that it is still gonadotropin. Even if there were a complete loss of activity of c-GTH at 100" in acetic acid 0.25% that could correspond to dissociation of the hormone (Burzawa-Gerard, 1971)) without complete destruction of the eventual subunits. Some reassociation could well occur during the elimination of acetic acid and concentration. Pituitary extracts from lungfish (Protopterus annectens) are very active on the spermiation in frogs (Burzawa-GBrard, 1969), and that is also the case for purified mammalian LH and HCG. LH, for instance, can be used as a routine standard but one has to be very cautious in calculating fish gonadotropin activity in terms of LH because the relative activities of these two hormones vary with the season (Burzawa-G&ard and Fontaine, 1965). In agreement with Meyer et al. (1961) we also find an action of FSH (Table 3) on frog spermiation, in the conditions of our assay. Other acute effects of gonadotropins on the testis have been used as criteria for bioassay. These effects are probably related to the ATPase activity depression, and to the swelling of the Sertoli cells observed by Burgos and Vitale (1968)) and Russo and Burgos (1969) under stimulation by LH or HCG. In larval Alytes obstetricans, carp pituitary extracts (Leray, 1963), purified c-GTH (BurzawaGerard and Fontaine, 1965), as well as NIH-LH and FSH (Delsol et al., 1970) at very low doses induce a rapid swelling of the testis. In juvenile Xenopus (Simon and Reinboth, 1966), extracts of fish pituitary, FSH, and LH determine a “vesicularisation reaction.” The relative activities of various substances on the different tests are summarized in Table 3. It is interesting to note that the activity
GONADOTROPINS
OF
VERTEBRATES
721
TABLE 3 ESTIXLTED REL..LTIVE ACTWITIES OF VARIOUS HORMOML PREP~IRATION~ ON SEVERAL AMPHIBIANS AND REPTILES 1 SPECIES
AUTHORS
NIH - FSH
G- GTH
CARPPtTUlTA~~1 I
+x
Delsolet al.
1970
BurzawaGerardand Fontaine 1965 SimonandReinboth1966
BtirzawaGerardand Fontaine 1965
1 (Sl-5 i2) 1 (Sl - $2) I
0.5
1 (83)
0.05
1 (Sl) 1
4.0 to
~~3)
i
_
-
-
12
0.8
%X% (Sl -
17
-
j
1
s5 1 '4
j carolinensis
i
austriaca i
kbt afldDonaldson 1969
Fontaineet al.
1971
1 (S6)
1
0.2
(S3)
(Sldl
-
0.06
-
-
-
/
-
i
“The activity of NIH- FSH is taken as 1 ““Number of the NIH preparation ***The activityof LH depends on the season.Thereis ROsignifica~ivedifferencebetweenthe activitiesof NIH - LH $5 and $9
ratio NIH-FSH/NIH-LH is very variable. If one remembers that NIH-LH is about 50% pure LH while NIH-FSH is only about 2% pure FSH, it appears that, in fact, FSH is more potent than LH (2500 x ) in all these tests. Histological studies on the action of FSH and LH on various aspect.s of testis function have been carried out by chronic (5-10 days) injections of rather high doses of hormones in hypophysectomised frogs. In these conditions LH appears to act mostly on the interstitial tissue and on spermiation while FSH clears the tubules of accumulated lipids, and stimulates the spermatogenesis (Burgos and Ladman, 19.57; Lofts, 1961; Van Oordt and De Kort, 1969). Recently, however, Redshaw and Nicholls (1971) have demonstrated an effect of PMSG on the biosynthesis of estrogens by Xenoptbs ovarian tissue. Recent information on the action of various gonadotropins on reptiles mainly
comes from the important works of Licht and his co-workers, who determined the testis conditions in Anolis carolinemis by several complementary methods. Licht and Donaldson (1969) found that 50 pg of G 100 one-GTH/day maintained the testis in their normal state after hypophysectomy, and promoted testicular growth and spermatogenesis in animals with naturally regressed gonads in the fall. Crude extracts of Anolis pituitaries stimulated both testicular growth and interstitial cell activity in hypophyseetomized animals (Licht. and Rosenberg, 1969). Finally, it was shown that both FSH and LH also stimmated the diverse aspects of testis function. The action of the two hormones is qualitatively similar but NIH-FSH is much more active than NIH-LH (Table 3). Interestingly enough if the experiments were made at, 20”, instead of the generally used temperature of activity is 31”, normal spermatogenetie
722
BURZAWA-GERARD
AND
FONTAINE
Gyclic AMP acsumulaied ( picomoles)
21.1 and 33.6
Controls
y&l qs Turtle e_GTH pituitary
ys NIH-LH Sll
‘Opll NMFSH s3
o.spm F Na
Addition per tube FIG. 3. HormonaI stimulation of cyclic AMP accumulation in a homogenate of ovary from CoroneEZu austriaea Laurenti. Cyclic AMP accumulation is determined according to Krishna et al. (1968). Incubation: 10 min at 20”. Pituitaries from turtles (Testudo radiata) of both sexes were extracted with a Tris HCl buffer pH 7.4., 0.05 M. Each column corresponds to one incubation tube.
best duplicated by a lower dose of gonadotropin than at 31” but this dose does not promote interstitial cell activity (Licht and Pearson, 1969b). In Hemiductylus, Reddy and Prasad (1970) have shown that FSH or PMSG, but not LH, stimulate both spermatogenesis and androgenic function. In another reptile, Agama agama, Eyeson (1971) observed that FSH and LH have qualitatively different effects, the first one stimulating spermatogenesis, and the second, interstitial tissue activity. With females somewhat similar results have been obtained. In Anolis, FSH and LH may also have the same qualitative effects but *FSH is much more potent than LH (Licht, 1970). In Lygosoma, Jones obtained positive results only with FSH while LH even seems to antagonize the
action of FSH (1969). We have tested the hormonal stimulation of cyclic AMP accumulation in an homogenate of ovary from a snake, Coronella austriaca. An extract of turtle pituitaries increases this accumulation. c-GTH has a low but significant action while NIH-FSH and NIH-LH have a very intense action (Fig. 3) (Fontaine et al., unpublished data). All these results show that amphibian and reptilian gonads exhibit the interesting property of being sensitive not only to their own GTH but also to both fish and mammal GTHs. The relative actions of these various hormones are generally quantitatively” and/or qualitatively different, 31t has to be noted that such differences observed in &DO may result from differences in the biological half-lives of the hormones.
GONADOTROPINS
according to the experimental animal ehosen and the external conditions. In mammals, finally, we were unable to produce any typical positive responses of the gonads with c-GTH (Burzawa-Gerard and Fontaine, 1965; Burzawa-Gerard, 1969). Moreover c-GTH (166 pg/ml) is inactive on adenyl cyclase activity of prepubertal rat ovary while 0.166 pg/ml NIH-LH or NIH-FSH are both active (Fontaine et al., 1971). Therefore c-GTH appears to be at. least 1000 times less active on this test than NIH-FSH or LH. These results lead to questions about the specificity of the positive effects observed years ago with generally crude materials by some workers (see Pickford and Ate, 1957). On the contrary some results indicate that GTH from lungfish (Barr and Hobson, 1966; Burzawa-Gerard, 1969), and amphibians (see Van Oordt and De Rort, 1969) can stimulate ovarian function in immature mice. DISCUSSIOP\T !L. Number of Gonadotropins Lower Vertebrates
in
The idea that two gonadotropins exist in lower vertebrates as in mammals has been favoured by most of the workers in this field until recently (see Pickford and Atz, 1957; Hoar, 1966). Three main arguments were responsible for that conclusion. (a) During their biological cycle many lower vertebrates exhibit a differential stimulation of gametogenetic and steroid-producing tissues, suggesting the existence of FSH-like and LH-like hormones (see Lofts, 1964; Van Oordt and De Kort, 1969; Hyder, 1970). However this hypothesis is not necessary. As regards amphibians for instance, Van Oordt and De Kort (1969) pointed out that “the cyclic changes in the frog’s testes can be explained on the basis of one gonadotropin and seasonal variation in sensitivity of the germinal epithelium and the interstitial tissue for gonadotropin.” Licht and Pearson (4969b) suggested that cyclic changes in :male reptiles can be explained by the variat,ions in the plasma level of one GTH
OF
VERTEBRATES
723
associated with a higher sensitivity in gametogenetic t,issue than in interstitial tissue. (b) The second argument derived from results obtained by injection of mammalian hormones into lower vertebrates. For instance the fact that LH but not FSH has sometimes been found active in fish led people to postulate the existence of a so called LH-like hormone in fish; obviously they were then also obliged to postulate a FSH-like hormone (see Hoar, 1966). In amphibians and reptiles mammalian FSH and LH can have different qualitative effects (Lofts, 1961; Eyeson, 1971), and this generally, here also, led one to think of endogenous FSR and LIT. However, even if these effects are direct (as suggested, in the case of the action of LH on teleost, testes by in vitro results of Wiebe, 1969), and not, due to some heterotropic action, that does not prove in our opinion, that there are actually two endogenous gonadotropins. What such interest’ing results demonstrate is rather that there are different hormona1 receptors with different specificities. (c) Two types of pituitary cells? both ha-c@ related to gonedotropic function, been described in many lower vertebrates, and have been then supposed to produce, respectively, a FSH-Iike and a LH-like hormone (see Hoar, 1966 ; Saint-Girons, 1967; Van Oordt, 1968). Without discussing this question we shal1 only poi.nt out that several discording reports appeared in recent years7 for instance that of Van Overbeeke and Mac Bride (1967j) Leatherland (1969), Mattheij (1970) in teleoste, and that of Van Kemenade (1969’) and Larsen et al. (1971) in amphibians. In Anolis LH cells are supposed to fined to the rostra1 region of the pars distalis. However, Licht and Pearson (1969a) found the same qualitative effect of partial hypophysectomy whatever region of the pars dist.alis was removed Moreover, injections of extracts of either caudal or rostra1 regions of the &and in hypophysectomized animals had similar quantitative and qualitative effects, both gametogenetic and interstitial tissue being
724
BURZAWA-GERARD
stimulated (Licht and Rosenberg, 1969). The cells previously identified as luteotropes may represent the corticotropes in reptiles (Licht and Bradshaw, 1969). In fact, it seems that the conception of two gonadotropins in lower vertebrates mainly comes from the fact that this duality had been demonstrated in mammals. As it is well-known, that particular conclusion was only achieved when it was possible to separate two distinct gonadotropic proteins, Then, on the contrary, fractionation of carp pituitary gave us only one fraction with gonadotropic activity and this result led us to consider seriously the possibility of only a single gonadotropin in this fish. Indeed this possibility was strongly suggested by the fact that the gonadotropic activity of carp pituitary, measured by various tests, was always present in the same purified c-GTH (Burzawa-Gerard and Fontaine, 1965). Later work, which has been summarized above, confirmed and extended this hypothesis: purified GTH from another teleost, Oncorhynchus, and also c-GTH, can normalize many very different aspects of gonadal function in hypophysectornized fish. The eventual generalization of the hypothesis concerning other lower vertebrates has to await until purification of GTHs. However we have seen that various recent results obtained in amphibians and reptiles agree with the idea of the unicity of GTH, while evidence against it is not, in our opinion, convincing. We believe that the existence of a single gonadotropin in lower vertebrates, even not, actually proven, should be now considered as a probable hypothesis. B. Specificity and Evolution Gonadotropin-s
of
Within teleost fish, a very polymorphous group indeed, there is an immunological and perhaps biological specificity (Pickford and Atz, 1957; Clemens and Johnson, 1964; Breton et al., unpublished data). Most information on the comparative properties of GTH from various classes of vertebrates is concerned with teleost and mammal hormones. It is clear that c-GTH
AND
FONTAINE
is biochemically quite different from mammalian FSH and LH. Immunological differences are also quite striking. Finally, most of the results so far obtained (and specially that on ovarian adenyl cyclase) lead one to emphasize the differences between the biological properties of teleost and mammal GTHs. However, studies in amphibians and reptiles have shown that both teleost and mammal GTHs can stimulate the gonads of these recipients. The existence of this common property, associated with a common glycoproteic nature and similar molecular weights suggest that, in spite of their differences, teleost and mammal gonadotropins are homologous proteins as is probabIy the case for TSHs (Fontaine, 1969c). Because mammalian TSHs and gonadotropins are also homologous (F’ontaine, 1969b; Pierce et al., 1971), GTHs and TSHs from all vertebrates probably belong to a same family of proteins. However we obviously need much more informationspecially on amphibian and reptilian GTHs-in order to define the evolution of these pituitary glycoproteins. The demonstration of stimulation of gonadal adenyl cyclase in teleost by teleost GTH as well as by FSH and LH in mammals suggests that the mechanism of action of GTH could be similar and follow the model of Sutherland (1968) in the various vertebrates. Then hormonal receptors for GTHs would be analogous in all the phyla and could be compared. The specificity of these receptors is indeed very variable. It is rather strict in some teleosts and in mammals ; on the contrary amphibian and reptilian gonadal receptors are able to recognize both piscine and mammalian GTHs (especially FSH), and look, therefore, intermediary between those existing in fish and in mammals. The specificity often appears also different from one species to another, for instance in amphibians or reptiles. Finally, some results suggest that even within one species the specificity of the gonadal receptors may be variable and depend upou, for instance, the sex (stimulation of eel gonads with mammalian gonadotropins; see Fontaine et al., 1966), the time of differentiation of the
OF
GONADOTROPINS
gonad (action of FSH on the amphibian Ceratophrys, Pisano and Burgos, 1971)) the season, or the temperature. These properties are reminiscent of that precedently found, in the case of the action of TSH and mammalian gonadotropins on thyroid, not in amphibians and reptiles, but in teleost fish (Fontaine, 196910). Both groups of results suggest that hormonal receptors implied in adenyl cyclase stimulation can have, specially in some lower vertebrates, a quite interesting plasticity. In conclusion, we would like to propose the following hypothesis, as a stimulus for discussion. In teleosts, and possibly also in amphibians and reptiles, a single pituitary gonadotropin is able to bind to the two classes of gonadal receptors, respectively in gametogenetic and steroid-producing tissues. A precise pituitary regulation is, however, achieved by a differentiation of these two classes of receptors. This differentiation can be concerned with t’he specificity of the receptors toward one part or another of t,he molecule of GTH or with the affinity of the receptors for GTH. It can even deal with the regulation of the recept,ors themselves, by other hormones for instance, a phenomenon which has been observed in other systems (Braun and Hechter, 1970). At some step-or steps-in the evolution, a new regulatory mechanism appears, a second pituitary hormone being brought into play with a division of the work between the two GTHs then existing. ACKNOWLEDGMENTS We ihank Dr. J. Marchelidon very much for performing amino arids determination. Standard preparations of FSI-I and LH have been kindly furnished by The Endocrinology Study Section (XIH). We are grateful to Drs. Breton, Billard, and Jalabert for communication of unpublished data, and to Drs. Parvez and Bradshaw for their help in correcting the English. We also thank Pr. J Guib& and his collaborators, Drs. F. d’Aubenton and M. Thireau for the gift of some amphibians and reptiles, and for their helpful advice. REFEREXCES =~HSAN,
zoo1
S.
41,
N.,
AND
1045-1053.
HOAR,
W.
S.
(1963).
Can.
.Z.
VERTEBRATES
BARR,
W.
A.,
AND
Endocrinol. BARR, W. A., .~ND Comp. Endocrinol. BILLARD,
(1970). BRAUN,
Acad. BRETON,
R.,
BILLARD,
4,
HOBSON,
R.
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FONTAINE, Y. A. (1969c). In “Proceedings of the Third International Congress of Endocrinology” (C. Gual, ed.), pp. 453-457. FONTAINE, Y. A., BURZAWA-GERARD, E., AND DELERUE-LEBELLE, N. (1970). 6. R. Acad. Sci. 271, 780-783. FONTAINE, Y. A., FONTAINE-BERTRAND, E., SALMON, C., AND DELERUE-LEBELLE, N. (1971). C. R. Acad. 8%. 2,72, 1137-1140. FONTAINE, Y. A., FONTAINE-BERTRAND, E., AND SALMON, C. Unpublished data. FONTAINE, Y. A., AND GERARD, E. (1963). C. R. Acad. Xci. 256, 5634-5637. GESCHWIND, I. I. (1969). Gen. Comp. Endocrinol. Suppl. 2, 180-189. GOSWAMI, S. V., AND SUNDARARAJ, B. I. (1968). Gen. Camp. EndocrinoE. 11, 393400. HOAR, W. S. (1966). In “The Pituitary Gland” (G. W. Harris and B. T. Donovan, eds.), pp. 242294. Butterworths. HOBSON, B. M., AND BARR, W. A. (1966). Gen. Comp. Endocrinol. 5, 686 (Abstr.). HYDER, M. (1970). Gen. Comp. Endocrinol. 14, 198211. HYDER, M., SHAH, A. V., AND KIRSCHNER, M. A. (1970). Endocrinology 87, 819-822. JONES, R. E. (1969). J. Ezp. Zool. 171, 217-221. JUTISZ, M., AND DE LA LLOSA, P. (1969). Colloq. Znt. Cent. Nat. Rech. Sci. 177, 293-314. KEMENADE, 5. A. M. VAN (1969). Z. Zellforsch. 96, 466-477. KIHLSTROM, J. E., AND DANNINGE, I. (1970). Gen. Comp. Endocrinol. 14, 592-593. KRISHNA, G., WEISS, B., AND BRODIE, B. B. (1968). J. Pharmacol. Exp. Therap. 163, 379-385. LARSEN, L. O., KEMENADE, J. A. M. VAN, AND DONGEN, W. J. VAN (1971). Gen. Comp. Endocrinol. 16, 165168. LEATHERLAND, J. F. (1969). Z. Zellforsch. 98,
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AND PEARSON, A. K. (1969b). Gen. Endocrinol. 13, 367381. P., AND ROSENBERG, L. L. (1969). Gen. Endocrinol. 13, 439-454. N. R., AND DONALDSON, E. M. (1969). J. Zool. 47, 569-573. B. (1961). Gen. Comp. Endocrinol. 1,
17!+189. LOFTS, B. (1964). Gen. Comp. Endocrinol. 4, 550-562. MATTHEIJ, J. A. M. (1970). Z. ZeZZjorsch. 105, 91-106. MENON, K. M. J., SMITH, M. (1971). Biochemktry 10, 1186-1190. MEYER, R. K., LEONORA, J., AND MC SHAN, W. H. (1961). Zn “Human Pituitary Gonadotropins” (A. Albert, ed.), pp. 326-328. C. C. Thomas Publishing. NAYYAR, S. K., AND SUNDARARAJ, B. I. (1969). J. Exp. Zool. 172, 385-398. OORDT, P. G. W. J. VAN (1968). In “Perspectives in Endocrinology: Hormones in the Lives of Vertebrates” (E. J. W. Barrington and C. B. Jorgensen, eds.), pp. 405467. Academic Press. OORDT, P. G. W. J. VAN, AND DE KORT, E. J. M. (1969). Colloq. Znt. Cent. Nat. Rech. Sci. 177, 345350. OVERBEEKE, A. P. VAN, MAC BRIDE, J. R. (1967). J. Fish. Res. Bd. Can. 24, 1791-1810. PAPKOFF, H., AND EKBLAD, M. (1970). Biochem. Biophys. Res. Comm. 40, 614-621. PICKFORD, G. E., AND ATZ, J. W. (1957). “The physiology of the pituitary gland of fishes,” 613 pp. New-York Zoological Sot., New York. PIERCE, J. G., LIAO, T. H., HOWARD, S. M., SHOMBE, B., AND CORNELL, J. S. (1971). Rec. Progr. Horm. Res. 27, 165-212. PISANO, A., AND BURGOS, M. H. (1971). Gen. Comp. Endocrinol. 16, 17&182. RAMASWAMI, L. S. (1962). Gen. Comp. Endocrinol. Suppl. 1, 286-299. REDDY, P. R. K., AND PRASAD, M. R. N. (1970). Gen. Comp. Endocrinol. 14, lCL.24. REDSHAW, M. R., AND NICHOLLS, T. J. (1971). Gen. Comp. Endocrinol. 16, 8696. REICHERT, L. E., RASCO, M. A., WARD, D. N., NISWENDER, G. D., AND MIDGLEY, A. R. (1969). J. Biol. Chem. 244, 5110-5117. Russo, J., AND BURGOS, M. H. (1969). Gen. Comp. Endocrinol. 13, 185-188. SAINT-GIRCINS, H. (1967). Ann. Sci. ,Nat. Zoot. Paris 9, 2229308. SIMON, N., AND REINBOTH, R. (1966). Verb. Deut. Zool. Ges. 3, Suppl. 254264. SUNDARARAJ, B. I., AND GOSWAMI, S. V. (1966a). J. Exp. Zool. 161, 287-296.
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163, 49-54. SUNDARARAJ, B. I., AND GOSWAMI, S. V. (1969). Gen. Camp. Endocrinol. Suppl. 2, 374-384. SUNDAR.~RAJ, B. I., AND NAYYAR, S. K. (1967). Gen. Comp. Endocrinol. 8, 403-416. SUTHEELAND, E. W., ROBEON, G. A., AND BUTCRER, R. W. (1968). Circulation 37, 279306. WARD,
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SWEENEY,
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VERTEBRATES
727
gress of Endocrinology” ilmsterdam, WIEBE: J. P. (1969). Gen. 267-275. WIEBE, J. YAMAZAKI,
Univ. YaMAZaKI,
Gen.
13,
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(1970).
F.
(1965). l-64.
F.,
Camp. YAMAZAKI, F., Gen. Camp. YAMAZAKI, F., Gen. Camp.
AND
(C.
Gual,
Camp.
Endocrinol. Mem. Fnc.
Doxa~~sox,
47, Fish. E.
ht.
383391. AND DONALDSOX, E. M. Endocrinol. 11, 292-299. AND
3$& 1
439. H&k& (~%%a).
16,
DONALDSON,
%ndocrinoI.
p
Er&~i~,o,ob
J.
Endocrinol.
ed.),
12,
E.
M.
491497.
DISCUSSION DONALDSON: Using salmon gonadotropin we have shown an effect on t,he uptake of radioactive phosphorous into the testis of the day-old male chick. In experiments in collaboration with Dr. Bern we have been unable to show any effect in the rat, and in our laboratory we have been unable to she-w any effect on ovarian or uterine weight in 21 day old female mouse. FONTAINE: Thank you. It is very interesting that one-GTH stimulates bird testis. From the point of view of the specificity of the gonadal response to GTH. that would suggest that birds are more similar to repti!es and amphibians ihan to mammals. GESCHWIND: I have two questions: (1) Is there any evidence of subunit structure in c-GTH? (2) Although c-GTH has no direct effect on adenyl cyclase activity in the rodent ovary, I wonder whether you have tried to use it in a competition experiment. that is, to determine whether c-GTH can inhibit the effect of ovine LH or FSH on the adenyl cyclase activity? This may prove to be a more sensitive test of a c-GTH effect in mammals. FONTAINE: There are indications of a subunit structure for c-GTH. After incubation of c-GTH at acid pH, Madame Burzawa observed that the sedimentation rate of the protein peak was much reduced, corresponding to a molecular weight around 15,000, instead of 30,000. On the other hand, if the purified c-GTH obtained by preparative electrophoresis and lyophilisation is again submitted to a preparative electrophoresis, two very well separated peaks appear. One corresponds to active c-GTH, the other to an inactive material with molecular weight around 15,000. The response to your second question is “no.” Indeed, we should try it. We have done some experiments of this kind in vivo. Some years ago Madame Burzawa observed that c-GTH injected with HCG (Steelman-Pohley test) did not inhibit the response to HCG. Also, there is no apparent inhibition of the effects of bovine TSH on mice by even high amounts of eel TSH. DONALDSON : At low pH, ultracentrifugation of salmon gonadotropin indicates a molecular weight of 13.700 which is similar to the value for carp gonadotropin. LICIIT: Have you studied the TSH activity of the GTH in any assay, i.e.: is there a possibility of a single glycoprotein with both activities? FONTAINE: There is evidence for the existence in teleosts of two different glycoproteins having thyrotropic or gonadotropic activity. For instance, the ratio of the GTH activity over the TSH activity can be very high in some species (carp). and low in others (eel). Also, this ratio increased along with t,he purification of c-GTH because the TSH and GTH peaks do not coincide in. Sephadex G 166 filtration, and DEAE-C chromatography. Thyrotropic activity of e-GTH after DEAE-C corresponded to a contamination by 4% c-TSH (w/w). We did not determine the TSH activity of c-GTH after the preparative electrophoresis because we would have had to use a relatively large amount of this precious material.
(19 (3.9699.
728
BURZAWA-GERARD
AND
FONTAINE
Finally, there are many examples of an independent physiological stimulation of gonads or thyroid. NICOLL: The recent data on the pituitary of poikilothermie vertebrates indicate the existence of only one gonadotropin, whereas the homeotherms evidently have two. The obvious question which arises is why mammals and birds need an addirelatives manage with one. Dr. tional gonadotropin when their “cold-blooded” Licht has emphasized the important effect of temperature in causing differential responsiveness of the gametogenic and steroidogenic activities of the gonads of poikilotherms. Perhaps the homeotherms need the second gonadotropin because they have lost the “benefit” of temperature fluctuations. Alternatively, the homeotherms may need two gonadotropins because they require more precisely timed ovulation in relation to insemination since their gametes have a much shorter life-span than those of the poikilotherms. WIEBE: In your hypothesis of specificity of various receptors to gonadotropins are you suggesting that the sensitivity of receptors varies seasonally as for example in cases where gametogenesis occurs before steroidogenesis? Is it perhaps also possible that there are two types of receptors, one for gonadotropins and one for steroids produced as a result of gonadotropin stimulation? In other words, is it possible to explain some of your results on the basis of a gonadotropin-receptor for steroidogenesis, and gametogenesis may then be the result of the gonadotropininduced steroidogenesis? FONTAINE: Indeed, the probable existence of a single pituitary gonadotropin in some lower vertebrates leads one to question the evolutionary significance of the second hormone. We suggest that an increasing differentiation of various classes of gonadal receptors could be important with regard to the appearance of a second gonadotropin. This phenomenon was also probably related to many other evolutionary changes, at a physiological level, such as those you suggest in your very interesting comment.
AND
COMPARATIVE
ENDOCRINOLOGY
SUPPLEMENT
The Gonadotropins
de Physiologie laboratoire
715-728
of lower
E. BURZAWA-GERARD Laboratoire nature&;
3,
AND
(197%
Vertebrates1
Y. A. FONTAINE
ge’ne’rale et compare’e du Mu&urn d%ndocrinologie compare’e associe’ Y, rue Cuvier, Paris 5e, France
national d’llistoire au C.NR.S.,
After reviewing data on comparative biochemical and biological properties of gonadotropins (GTH) from lower vertebrates, we discuss two main problems: the number of GTHs in each lower vertebrate, and the specificity and evolutioa of GTHs. It is concluded that: (1) The existence of a single GTN in the teleost, and possibly also in the amphibian and reptile, is the most probable hypothesis. (2) While all GTHs (and TSHs) are probably homologous proteins, an important zoological specificity of GTH does exist. Enally, preliminary results suggest that the gonadal receptors for GTH in iowcr vertebrates are, as in mammals, associated with an adenyl cyclase activi;ily. The evolution of these “analogous” receptors should be considered together with that of the gonadotropins. It is suggested that the differentiation of the two classes of receptors (respectively, in gametogenetic and steroid-producing tissues) allowed a precise pituitary regulation of the gonads before the appearance of a second pituitary gonadotropin.
In all lower vertebrates, as in mammals, the gonads are under the dependence of the pituitary, even if this dependence can be less rigid in some groups, Cyclostomes for instance (see Pickford and Ate, 1957; Dodd, 1960; Fontaine, 1968; Licht, and Pearson, 1969a). This control is exerted, at least principally, by hormones which act directly upon gonadal tissue, and which are synthesized and secreted by specialized cells of the anterior pituitary. The present paper will deal with these gonadotropin hormones (GTH) but it will not be an exhaustive review due to the very large amount of information avail-
able. For instance? the eventual interference of other hormones (~~prola~t~~~‘~ steroids, etc.) in the action of be left aside. We shall main two problems: number of GT lower vertebrate, and specificity and evolution, of GTHs. In order to discuss these points we shall first describe some recent results concerning the p~r~fi~at~~~ of lower vertebrate GTHs, and the comparison of biochemical and b~~l~~i~al properties of GTHR from various vertebrates.
’ GTH : gonadotropic hormone ; LH : luteinizing hormone; FSH: follicle-stimulating hormone ; TSH: thyrotropic hormone ; HCG : human ehorionic gonadotropin; PMSG : pregnant mare serum gonadotropin. A letter, or letters preceding the name of a hormone indicates the zoological origin by referring to the latin genus name of the species considered: e.g., c = Cypri32215, o = ovine, one = Oncorhynchus; DEAE-C = diethylaminoethyl cellulose ; AMP = adenosine monophosphate. KD = exclusion coefficient on Sephadex.
So far only teleost G purified. Because, as we shall see, these hormones do not have t,ypical actions in mammals, bioassays have to be made on lower vertebrates.
I-PURIFICATION FROM
by
Academic
Press,
Inc.
GONADOTROPINS, VERTEBRATES
A. Purification of c-GTlrl This purification has been followed by measuring the gonadotropic activity frog spermiation in the conditions scribed by Fontaine and Chauvel (1961). 715
@ 1972
OF LOWER
716
BURZAWA-GERARD
AND
The first steps of the purification, which have been described in detail elsewhere (Fontaine and G&ard, 1963) are summarized in Fig. 1. The recent addition of a preparative electrophoresis on polyacrylamide gel led to a better purification (Buraawa-GBrard, 1971). Figure 2 shows that the biologically active peak is eluted with an Rf of about 0.50, and separated from an inactive material (Rf = 0.80). After recovery on Sephadex G 100, the lyophilised powder is about 20 times more active than the starting material. Its activity is 1.5 X NIH LH Sl (determination made in October). The yield in activity is about 40%. Another attempt to prepare c-GTH has been made by Clemens et al. (1964). They followed the’activity by measuring gonadal hydration in goldfish (Clemens and Grant, 1964), and used only a Sephadex G 75
FONTAINE
filtration. In spite of some apparent discrepancy it seems probable that the material they obtained corresponds to t;he same c-GTH we purified (BurzawaGlBrard and -Fontaine, 1966). B. Purification of one-GTH Donaldson and Yamazaki (1968) have purified a GTH from pituitaries of Pacific Salmon (Oncorhynchus tshawytscha) . They followed the activity by studying the induction of spermiation in hypophysectomised goldfish (Yamazaki and Donaldson, 1968a). The biochemical procedure does not seem to have been published in detail; these authors used an alcoholic extraction followed by two filtrations on Sephadex G 100, and eventually DEAE-C chromatography. The partly purified oncGTH (after G 100) had an activity of 3500
Extraction‘by AlcoholicPercolation I
1
Fractionsolohle in ( Et OH57% NaCl 2 %)
Unretardedfraction
1fi~:!wf~.“:‘“‘- C1 Adsorhedfraction eluted by gradient of ionic strenghth
Fraction of KD = 0.28 Preparative electrophoresis Fraction of RF
=
0.50 -0.54
Fractionof KD= 0.28 Lyophylisation---+x4-1 FIG. 1. Main
steps
in the purification
of c-GTH.
GONADOTROPINS
OF
717
VERTEBRATES
t-5~o~o~icalactivity in termsof NH- LHI- SIT
0
50
100
150
Effluent(ml)
FIG. 2. Preparative electrophoresis of partly purified c-GTH (after Sephsdex G 100 and I?EAE-C) on polyacrylamide gel. O-O Proteins (o.d. 276 mp). 0-0 Biological activity, measured cr, frog spermislion test, in terms of NIH-LH-Sll (pg/mt). ? -No animal responds to the inje&ed dose. 8 --All animals -respond to the injected dose. During the electrophoresis the material is concentrated in the upper gel (2.57; acrylamide), and the individual components are separated in Tris-Glycine buffer (Tris 0.36 &f, Giycine 0.043 ilf ; pH 10.3) in the resolving gel (7yc acrylamide). The migration occurs from the anode to the cathode and the proteins are &ted in Tris-WC1 bufYer pH 8.1 (0.1 Ai Tris). I .= 40 mA, V = 200 V, and el!llion rate 10 ml/hour.
IU HCG/mg (HCG being active in the bioassay can be used as a standard). II-BIOCHEMICAL PROPERTIES OF WITH MAMMALIAN
AND IMMUNOLOGICAL c-GTH. COMPARISON GONADOTROPINS
The behaviour of the biological activity on ion exchangers, and the electrophoretical properties of purified e-GTH show that it is rather an acidic protein like mammalian FSHs. The molecular weight of c-GTH has been shown by sucrose gradient centrifugation to be around 27,000-31,000 (Burzawa-Gitrard and Fontaine, 1966 ; Burzawa-C&ard, 1971) very close to that of mammalian gonadotropins (see Geschwind, 1969; Jut&z and De la Llosa, 1969). The homogeneity of purified c-GTH has
been studied by ana,lytical and preparative electrophoresis. If, for instance, the c-GTH obtained by lyophilisation of the aclive peak (RP = 0.50) in preparative electrophoresis is subjected to anot,her preparative electrophoresis, again two proteie peaks are observed, one active (Rf = O.SO), and one inactive (Idl = 0.80). The maleeular weight of this last materiaE is about half of that. of the active protein (Burzawa-G&ard? 1971). This result sugges-& that c-GTH might, be dissociable in t,wo subunits as it has been shown for mammalian LHs (see Jutisz and De la Llqsa, 1969; Geschwind, 1969), TSHs (Pierce et aE., 19X), and finally FSHs @q&off and Ekblad, 1970). Amino acid composition of c-GTH has been determined and Table I
718
BURZAWA-GERARD
COMPOSITION
OF c-GTH
AND
AND
FONTAINE
TABLE 1 COMPARISON WITH THAT
comparison with the compositions of ovine LH and FSH. It is clear that c-GTH is quite different from both mammalian FSHs and LHs. Finally, c-GTH appears to be a glycoprotein like mammalian GTHs (Table 1). c-GTH is antigenic in guinea pigs, or rabbits. Antiserum obtained from rabbits immunised against c-GTH gives in immunoelectrophoresis two precipitation arcs which probably correspond to the proteins having Rf of, respectively, 0.50 and 0.80 in acrylamide electrophoresis. On the contrary there is no precipitation between the same antiserum and o-LH or o-FSH. By use of antibodies obtained in guinea pigs, and of a labeled 13?I c-GTH, a radioimmunoassay of c-GTH has recently been established. In this system there appears to be some cross reaction bet,ween C-GTH and o-LH (Breton et .aZ.j 1971).
OF MAMMALIAN
FSH
AND
LHs
III-COMPARISON OF THE BIOLOGICAL PROPERTIES OF VARIOUS GONADOTROPINS
A. On Teleost Fish c-GTH is actually secreted in the blood as shown by radioimmunoassay (Breton et al., unpublished data). It stimulates many parameters of the gonadal function in teleost fish: hydration of the testis,2 32P uptake by eel testis (Buwawa-Gerard and Fontaine, 1965), adenyl cyclase activity in goldfish at the beginning of the vitellogenesis (Fontaine et al., 1970). By these various quantitative tests, the activity ratio c-GTH/carp pituitary is statisaWhen testing electrophoresis by activity is in frog spermiation
the effluent of a preparative this method, the maximum the same tubes as by use of the test.
GONADOTROPINS
OF
tically the same as determined in amphibians (Table 2). In hypophysectomieed goldfish, e-GTH (1 pg/g) restores spermatogenesis and spermiation in males (Billard et al., 19?0), and vitellogenesis in females (Breton et al., unpublished dat’a) . These last results agree very well with those obtained with purified one-GTH by Yamazaki and Donaldson (1968b) who, moreover, showed an action on the ovulation, and a stimulation of the act.ivity of the 3 P-ol-dehydrogenase (1969). Liley and Donaldson (1969) observed with oncGTH, stimulation of growth and maturation of the ovary in the guppy (Poecilia reticulata), and their results also suggest a stimulation of the production of an ovarian hormone involved in the regulation of sexual behavior. Menon and Smith (1971) observed an increase cyclic AMP accumulation in slices of testis from chinook salmon under the influence of one-GTH. Studies of the effects on fish of gonadotropic preparations from pituitaries of RATIO
Species
Reference
BurzawaGerard and Fontaine1965
C-GTHJCARP
FISH Anguilla anguilla Carassiusauratus
AMPHIBIANS BurzawaGirard andFontaine$965 Gurzawa Gerard1971 Ranaesculenta Hohsonand Barr1966 _ idem_ BurzawaGerard andFontaine1985
PITUITARY
2 MEASURED
Test
ON VARIOUS
RECIPIEXTS
Second preparatiea ( BurzawaGerard 1971)
First preparation (FontaineandGsrard 1963)
11.6 *
32P uptakebythe testis (4.4
Fontaineandal.rno
n.9
amphibian and reptiles gave conflicting results. The same is true with mammalia,n LH, while mammalian FSH is generally inactive (see Pickford and Atz, 1957; Hoar, 1966; Sundararaj and Nayyar, 1967; Goswami and Sundararaj, 1968). The stimulation of various aspects of gonadxi activity by LH has been observed for instance by Ashen and Hoar (1963)) Yamazaki (1965), Wiebe (1969)) but LH has no action on spermiation (Yamazaki and Donaldson, 1968a) ) or on spermatogenesis (Billard et al., 1970) in the hypophysectomized goldfish. In quantitative bioassays (hydration of the testis, 32P testis uptake) mammalian gonadotropins have either no action, or a low non-typical action (which does not increase with the dose) (Clemens and Johnson, 1964; Burzawa-G,Brard and Fontaine, 1965 ; Burzawa-Gerard, 1969) _ No action of mammalian LH and FSH is seen on goldfish ovarian adenyl cyclase either in quiescent ovaries or at t.he end of the spa-wning, while c-GTE is active in both cases (Fontaine et al., 197%; I?ontaine et ai., unpublished data). Positive
TABLE ACTIVITY
VERTEBRATES
- 30.3)
20
ovarianadenylcyclase
Spermiation
6.8 (4.3
5ana pipiens*++
Spermiation
6.6
Xenopuslaevis’*
Spermiation
11.2 7.4”
/ Alytesobstetricans Swellingof the testis (0.23
15 *:
*
- 10.7)
(Ia
1
- 41.7) I
* Limits for p= 0.95
=**Kindly testedby 5 Barr
- 22 )
720
BURZAWA-GERARD
results have been more consistently obtained with human chorionic gonadotropin. HCG determines the maturation of gonads in male eels (Fontaine, 1936) but not in female eels where c-GTH does so (Fontaine et al., 1964; Burzawa-Gerard, 1969). In other species of teleosts HCG is active on various aspects of the gonadal function, both in males (Sundararaj and Nayyar, 1967; Yamazaki and Donaldson, 1968a; Hyder et al., 1970)) and in females (Yamazaki, 1965; Goswami and Sundararaj, 1968). Mammalian gonadotropins might stimulate some aspects of gonadal function not directly but by the intermediary of a heterotropic action. It is known that mammalian gonadotropins can stimulate teleost thyroids (Fontaine, 1969a). Similarly, papers from Sundararaj and Goswami (1966a and b, 1969)) and Nayyar and Sundararaj (1969) show that LH acts upon the secretory activity of the interrenal in the catfish; in turn corticosteroids would be responsible for the observed action of LH on ovulation and spawning, as previously shown (see Ramaswami, 1962). B. On Amphibians,
Reptiles, and
Mammals
As far as the amphibians are concerned we shall limit ourselves to the studies done on males. Extracts of teleost pituitaries can induce spermiation in the frogs Rana esculenta (Fontaine and Chauvel, 1961), and Xenopus Zaewis (Barr and Hobson, 1964). During the fractionation of carp pituitary by various biochemical methods the activity was followed by the frog spermiation test. In any case only one active peak was found. Our results suggest that in carp pituitary only the carp gonadotropin c-GTH is active on frog spermiation at least in the conditions described by Fontaine and Chauvel (1961). Kihlstrom and Danninge (1970) have suggested that this response can be elicited by factors present in frog pituitary which are not the gonadotropins on the basis of the positive results they obtained after
AND
FONTAINE
extraction of frog pituitaries at 100” by acetic acid 0.25%, and evaporation. Indeed we have obtained somewhat similar results with carp pituitary; however quantitatively only 10% of the original activity is present after this treatment; the nature of this active material has to be investigated further but it does not appear impossible that it is still gonadotropin. Even if there were a complete loss of activity of c-GTH at 100" in acetic acid 0.25% that could correspond to dissociation of the hormone (Burzawa-Gerard, 1971)) without complete destruction of the eventual subunits. Some reassociation could well occur during the elimination of acetic acid and concentration. Pituitary extracts from lungfish (Protopterus annectens) are very active on the spermiation in frogs (Burzawa-GBrard, 1969), and that is also the case for purified mammalian LH and HCG. LH, for instance, can be used as a routine standard but one has to be very cautious in calculating fish gonadotropin activity in terms of LH because the relative activities of these two hormones vary with the season (Burzawa-G&ard and Fontaine, 1965). In agreement with Meyer et al. (1961) we also find an action of FSH (Table 3) on frog spermiation, in the conditions of our assay. Other acute effects of gonadotropins on the testis have been used as criteria for bioassay. These effects are probably related to the ATPase activity depression, and to the swelling of the Sertoli cells observed by Burgos and Vitale (1968)) and Russo and Burgos (1969) under stimulation by LH or HCG. In larval Alytes obstetricans, carp pituitary extracts (Leray, 1963), purified c-GTH (BurzawaGerard and Fontaine, 1965), as well as NIH-LH and FSH (Delsol et al., 1970) at very low doses induce a rapid swelling of the testis. In juvenile Xenopus (Simon and Reinboth, 1966), extracts of fish pituitary, FSH, and LH determine a “vesicularisation reaction.” The relative activities of various substances on the different tests are summarized in Table 3. It is interesting to note that the activity
GONADOTROPINS
OF
VERTEBRATES
721
TABLE 3 ESTIXLTED REL..LTIVE ACTWITIES OF VARIOUS HORMOML PREP~IRATION~ ON SEVERAL AMPHIBIANS AND REPTILES 1 SPECIES
AUTHORS
NIH - FSH
G- GTH
CARPPtTUlTA~~1 I
+x
Delsolet al.
1970
BurzawaGerardand Fontaine 1965 SimonandReinboth1966
BtirzawaGerardand Fontaine 1965
1 (Sl-5 i2) 1 (Sl - $2) I
0.5
1 (83)
0.05
1 (Sl) 1
4.0 to
~~3)
i
_
-
-
12
0.8
%X% (Sl -
17
-
j
1
s5 1 '4
j carolinensis
i
austriaca i
kbt afldDonaldson 1969
Fontaineet al.
1971
1 (S6)
1
0.2
(S3)
(Sldl
-
0.06
-
-
-
/
-
i
“The activity of NIH- FSH is taken as 1 ““Number of the NIH preparation ***The activityof LH depends on the season.Thereis ROsignifica~ivedifferencebetweenthe activitiesof NIH - LH $5 and $9
ratio NIH-FSH/NIH-LH is very variable. If one remembers that NIH-LH is about 50% pure LH while NIH-FSH is only about 2% pure FSH, it appears that, in fact, FSH is more potent than LH (2500 x ) in all these tests. Histological studies on the action of FSH and LH on various aspect.s of testis function have been carried out by chronic (5-10 days) injections of rather high doses of hormones in hypophysectomised frogs. In these conditions LH appears to act mostly on the interstitial tissue and on spermiation while FSH clears the tubules of accumulated lipids, and stimulates the spermatogenesis (Burgos and Ladman, 19.57; Lofts, 1961; Van Oordt and De Kort, 1969). Recently, however, Redshaw and Nicholls (1971) have demonstrated an effect of PMSG on the biosynthesis of estrogens by Xenoptbs ovarian tissue. Recent information on the action of various gonadotropins on reptiles mainly
comes from the important works of Licht and his co-workers, who determined the testis conditions in Anolis carolinemis by several complementary methods. Licht and Donaldson (1969) found that 50 pg of G 100 one-GTH/day maintained the testis in their normal state after hypophysectomy, and promoted testicular growth and spermatogenesis in animals with naturally regressed gonads in the fall. Crude extracts of Anolis pituitaries stimulated both testicular growth and interstitial cell activity in hypophyseetomized animals (Licht. and Rosenberg, 1969). Finally, it was shown that both FSH and LH also stimmated the diverse aspects of testis function. The action of the two hormones is qualitatively similar but NIH-FSH is much more active than NIH-LH (Table 3). Interestingly enough if the experiments were made at, 20”, instead of the generally used temperature of activity is 31”, normal spermatogenetie
722
BURZAWA-GERARD
AND
FONTAINE
Gyclic AMP acsumulaied ( picomoles)
21.1 and 33.6
Controls
y&l qs Turtle e_GTH pituitary
ys NIH-LH Sll
‘Opll NMFSH s3
o.spm F Na
Addition per tube FIG. 3. HormonaI stimulation of cyclic AMP accumulation in a homogenate of ovary from CoroneEZu austriaea Laurenti. Cyclic AMP accumulation is determined according to Krishna et al. (1968). Incubation: 10 min at 20”. Pituitaries from turtles (Testudo radiata) of both sexes were extracted with a Tris HCl buffer pH 7.4., 0.05 M. Each column corresponds to one incubation tube.
best duplicated by a lower dose of gonadotropin than at 31” but this dose does not promote interstitial cell activity (Licht and Pearson, 1969b). In Hemiductylus, Reddy and Prasad (1970) have shown that FSH or PMSG, but not LH, stimulate both spermatogenesis and androgenic function. In another reptile, Agama agama, Eyeson (1971) observed that FSH and LH have qualitatively different effects, the first one stimulating spermatogenesis, and the second, interstitial tissue activity. With females somewhat similar results have been obtained. In Anolis, FSH and LH may also have the same qualitative effects but *FSH is much more potent than LH (Licht, 1970). In Lygosoma, Jones obtained positive results only with FSH while LH even seems to antagonize the
action of FSH (1969). We have tested the hormonal stimulation of cyclic AMP accumulation in an homogenate of ovary from a snake, Coronella austriaca. An extract of turtle pituitaries increases this accumulation. c-GTH has a low but significant action while NIH-FSH and NIH-LH have a very intense action (Fig. 3) (Fontaine et al., unpublished data). All these results show that amphibian and reptilian gonads exhibit the interesting property of being sensitive not only to their own GTH but also to both fish and mammal GTHs. The relative actions of these various hormones are generally quantitatively” and/or qualitatively different, 31t has to be noted that such differences observed in &DO may result from differences in the biological half-lives of the hormones.
GONADOTROPINS
according to the experimental animal ehosen and the external conditions. In mammals, finally, we were unable to produce any typical positive responses of the gonads with c-GTH (Burzawa-Gerard and Fontaine, 1965; Burzawa-Gerard, 1969). Moreover c-GTH (166 pg/ml) is inactive on adenyl cyclase activity of prepubertal rat ovary while 0.166 pg/ml NIH-LH or NIH-FSH are both active (Fontaine et al., 1971). Therefore c-GTH appears to be at. least 1000 times less active on this test than NIH-FSH or LH. These results lead to questions about the specificity of the positive effects observed years ago with generally crude materials by some workers (see Pickford and Ate, 1957). On the contrary some results indicate that GTH from lungfish (Barr and Hobson, 1966; Burzawa-Gerard, 1969), and amphibians (see Van Oordt and De Rort, 1969) can stimulate ovarian function in immature mice. DISCUSSIOP\T !L. Number of Gonadotropins Lower Vertebrates
in
The idea that two gonadotropins exist in lower vertebrates as in mammals has been favoured by most of the workers in this field until recently (see Pickford and Atz, 1957; Hoar, 1966). Three main arguments were responsible for that conclusion. (a) During their biological cycle many lower vertebrates exhibit a differential stimulation of gametogenetic and steroid-producing tissues, suggesting the existence of FSH-like and LH-like hormones (see Lofts, 1964; Van Oordt and De Kort, 1969; Hyder, 1970). However this hypothesis is not necessary. As regards amphibians for instance, Van Oordt and De Kort (1969) pointed out that “the cyclic changes in the frog’s testes can be explained on the basis of one gonadotropin and seasonal variation in sensitivity of the germinal epithelium and the interstitial tissue for gonadotropin.” Licht and Pearson (4969b) suggested that cyclic changes in :male reptiles can be explained by the variat,ions in the plasma level of one GTH
OF
VERTEBRATES
723
associated with a higher sensitivity in gametogenetic t,issue than in interstitial tissue. (b) The second argument derived from results obtained by injection of mammalian hormones into lower vertebrates. For instance the fact that LH but not FSH has sometimes been found active in fish led people to postulate the existence of a so called LH-like hormone in fish; obviously they were then also obliged to postulate a FSH-like hormone (see Hoar, 1966). In amphibians and reptiles mammalian FSH and LH can have different qualitative effects (Lofts, 1961; Eyeson, 1971), and this generally, here also, led one to think of endogenous FSR and LIT. However, even if these effects are direct (as suggested, in the case of the action of LH on teleost, testes by in vitro results of Wiebe, 1969), and not, due to some heterotropic action, that does not prove in our opinion, that there are actually two endogenous gonadotropins. What such interest’ing results demonstrate is rather that there are different hormona1 receptors with different specificities. (c) Two types of pituitary cells? both ha-c@ related to gonedotropic function, been described in many lower vertebrates, and have been then supposed to produce, respectively, a FSH-Iike and a LH-like hormone (see Hoar, 1966 ; Saint-Girons, 1967; Van Oordt, 1968). Without discussing this question we shal1 only poi.nt out that several discording reports appeared in recent years7 for instance that of Van Overbeeke and Mac Bride (1967j) Leatherland (1969), Mattheij (1970) in teleoste, and that of Van Kemenade (1969’) and Larsen et al. (1971) in amphibians. In Anolis LH cells are supposed to fined to the rostra1 region of the pars distalis. However, Licht and Pearson (1969a) found the same qualitative effect of partial hypophysectomy whatever region of the pars dist.alis was removed Moreover, injections of extracts of either caudal or rostra1 regions of the &and in hypophysectomized animals had similar quantitative and qualitative effects, both gametogenetic and interstitial tissue being
724
BURZAWA-GERARD
stimulated (Licht and Rosenberg, 1969). The cells previously identified as luteotropes may represent the corticotropes in reptiles (Licht and Bradshaw, 1969). In fact, it seems that the conception of two gonadotropins in lower vertebrates mainly comes from the fact that this duality had been demonstrated in mammals. As it is well-known, that particular conclusion was only achieved when it was possible to separate two distinct gonadotropic proteins, Then, on the contrary, fractionation of carp pituitary gave us only one fraction with gonadotropic activity and this result led us to consider seriously the possibility of only a single gonadotropin in this fish. Indeed this possibility was strongly suggested by the fact that the gonadotropic activity of carp pituitary, measured by various tests, was always present in the same purified c-GTH (Burzawa-Gerard and Fontaine, 1965). Later work, which has been summarized above, confirmed and extended this hypothesis: purified GTH from another teleost, Oncorhynchus, and also c-GTH, can normalize many very different aspects of gonadal function in hypophysectornized fish. The eventual generalization of the hypothesis concerning other lower vertebrates has to await until purification of GTHs. However we have seen that various recent results obtained in amphibians and reptiles agree with the idea of the unicity of GTH, while evidence against it is not, in our opinion, convincing. We believe that the existence of a single gonadotropin in lower vertebrates, even not, actually proven, should be now considered as a probable hypothesis. B. Specificity and Evolution Gonadotropin-s
of
Within teleost fish, a very polymorphous group indeed, there is an immunological and perhaps biological specificity (Pickford and Atz, 1957; Clemens and Johnson, 1964; Breton et al., unpublished data). Most information on the comparative properties of GTH from various classes of vertebrates is concerned with teleost and mammal hormones. It is clear that c-GTH
AND
FONTAINE
is biochemically quite different from mammalian FSH and LH. Immunological differences are also quite striking. Finally, most of the results so far obtained (and specially that on ovarian adenyl cyclase) lead one to emphasize the differences between the biological properties of teleost and mammal GTHs. However, studies in amphibians and reptiles have shown that both teleost and mammal GTHs can stimulate the gonads of these recipients. The existence of this common property, associated with a common glycoproteic nature and similar molecular weights suggest that, in spite of their differences, teleost and mammal gonadotropins are homologous proteins as is probabIy the case for TSHs (Fontaine, 1969c). Because mammalian TSHs and gonadotropins are also homologous (F’ontaine, 1969b; Pierce et al., 1971), GTHs and TSHs from all vertebrates probably belong to a same family of proteins. However we obviously need much more informationspecially on amphibian and reptilian GTHs-in order to define the evolution of these pituitary glycoproteins. The demonstration of stimulation of gonadal adenyl cyclase in teleost by teleost GTH as well as by FSH and LH in mammals suggests that the mechanism of action of GTH could be similar and follow the model of Sutherland (1968) in the various vertebrates. Then hormonal receptors for GTHs would be analogous in all the phyla and could be compared. The specificity of these receptors is indeed very variable. It is rather strict in some teleosts and in mammals ; on the contrary amphibian and reptilian gonadal receptors are able to recognize both piscine and mammalian GTHs (especially FSH), and look, therefore, intermediary between those existing in fish and in mammals. The specificity often appears also different from one species to another, for instance in amphibians or reptiles. Finally, some results suggest that even within one species the specificity of the gonadal receptors may be variable and depend upou, for instance, the sex (stimulation of eel gonads with mammalian gonadotropins; see Fontaine et al., 1966), the time of differentiation of the
OF
GONADOTROPINS
gonad (action of FSH on the amphibian Ceratophrys, Pisano and Burgos, 1971)) the season, or the temperature. These properties are reminiscent of that precedently found, in the case of the action of TSH and mammalian gonadotropins on thyroid, not in amphibians and reptiles, but in teleost fish (Fontaine, 196910). Both groups of results suggest that hormonal receptors implied in adenyl cyclase stimulation can have, specially in some lower vertebrates, a quite interesting plasticity. In conclusion, we would like to propose the following hypothesis, as a stimulus for discussion. In teleosts, and possibly also in amphibians and reptiles, a single pituitary gonadotropin is able to bind to the two classes of gonadal receptors, respectively in gametogenetic and steroid-producing tissues. A precise pituitary regulation is, however, achieved by a differentiation of these two classes of receptors. This differentiation can be concerned with t’he specificity of the receptors toward one part or another of t,he molecule of GTH or with the affinity of the receptors for GTH. It can even deal with the regulation of the recept,ors themselves, by other hormones for instance, a phenomenon which has been observed in other systems (Braun and Hechter, 1970). At some step-or steps-in the evolution, a new regulatory mechanism appears, a second pituitary hormone being brought into play with a division of the work between the two GTHs then existing. ACKNOWLEDGMENTS We ihank Dr. J. Marchelidon very much for performing amino arids determination. Standard preparations of FSI-I and LH have been kindly furnished by The Endocrinology Study Section (XIH). We are grateful to Drs. Breton, Billard, and Jalabert for communication of unpublished data, and to Drs. Parvez and Bradshaw for their help in correcting the English. We also thank Pr. J Guib& and his collaborators, Drs. F. d’Aubenton and M. Thireau for the gift of some amphibians and reptiles, and for their helpful advice. REFEREXCES =~HSAN,
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Endocrinol.
ed.),
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DISCUSSION DONALDSON: Using salmon gonadotropin we have shown an effect on t,he uptake of radioactive phosphorous into the testis of the day-old male chick. In experiments in collaboration with Dr. Bern we have been unable to show any effect in the rat, and in our laboratory we have been unable to she-w any effect on ovarian or uterine weight in 21 day old female mouse. FONTAINE: Thank you. It is very interesting that one-GTH stimulates bird testis. From the point of view of the specificity of the gonadal response to GTH. that would suggest that birds are more similar to repti!es and amphibians ihan to mammals. GESCHWIND: I have two questions: (1) Is there any evidence of subunit structure in c-GTH? (2) Although c-GTH has no direct effect on adenyl cyclase activity in the rodent ovary, I wonder whether you have tried to use it in a competition experiment. that is, to determine whether c-GTH can inhibit the effect of ovine LH or FSH on the adenyl cyclase activity? This may prove to be a more sensitive test of a c-GTH effect in mammals. FONTAINE: There are indications of a subunit structure for c-GTH. After incubation of c-GTH at acid pH, Madame Burzawa observed that the sedimentation rate of the protein peak was much reduced, corresponding to a molecular weight around 15,000, instead of 30,000. On the other hand, if the purified c-GTH obtained by preparative electrophoresis and lyophilisation is again submitted to a preparative electrophoresis, two very well separated peaks appear. One corresponds to active c-GTH, the other to an inactive material with molecular weight around 15,000. The response to your second question is “no.” Indeed, we should try it. We have done some experiments of this kind in vivo. Some years ago Madame Burzawa observed that c-GTH injected with HCG (Steelman-Pohley test) did not inhibit the response to HCG. Also, there is no apparent inhibition of the effects of bovine TSH on mice by even high amounts of eel TSH. DONALDSON : At low pH, ultracentrifugation of salmon gonadotropin indicates a molecular weight of 13.700 which is similar to the value for carp gonadotropin. LICIIT: Have you studied the TSH activity of the GTH in any assay, i.e.: is there a possibility of a single glycoprotein with both activities? FONTAINE: There is evidence for the existence in teleosts of two different glycoproteins having thyrotropic or gonadotropic activity. For instance, the ratio of the GTH activity over the TSH activity can be very high in some species (carp). and low in others (eel). Also, this ratio increased along with t,he purification of c-GTH because the TSH and GTH peaks do not coincide in. Sephadex G 166 filtration, and DEAE-C chromatography. Thyrotropic activity of e-GTH after DEAE-C corresponded to a contamination by 4% c-TSH (w/w). We did not determine the TSH activity of c-GTH after the preparative electrophoresis because we would have had to use a relatively large amount of this precious material.
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Finally, there are many examples of an independent physiological stimulation of gonads or thyroid. NICOLL: The recent data on the pituitary of poikilothermie vertebrates indicate the existence of only one gonadotropin, whereas the homeotherms evidently have two. The obvious question which arises is why mammals and birds need an addirelatives manage with one. Dr. tional gonadotropin when their “cold-blooded” Licht has emphasized the important effect of temperature in causing differential responsiveness of the gametogenic and steroidogenic activities of the gonads of poikilotherms. Perhaps the homeotherms need the second gonadotropin because they have lost the “benefit” of temperature fluctuations. Alternatively, the homeotherms may need two gonadotropins because they require more precisely timed ovulation in relation to insemination since their gametes have a much shorter life-span than those of the poikilotherms. WIEBE: In your hypothesis of specificity of various receptors to gonadotropins are you suggesting that the sensitivity of receptors varies seasonally as for example in cases where gametogenesis occurs before steroidogenesis? Is it perhaps also possible that there are two types of receptors, one for gonadotropins and one for steroids produced as a result of gonadotropin stimulation? In other words, is it possible to explain some of your results on the basis of a gonadotropin-receptor for steroidogenesis, and gametogenesis may then be the result of the gonadotropininduced steroidogenesis? FONTAINE: Indeed, the probable existence of a single pituitary gonadotropin in some lower vertebrates leads one to question the evolutionary significance of the second hormone. We suggest that an increasing differentiation of various classes of gonadal receptors could be important with regard to the appearance of a second gonadotropin. This phenomenon was also probably related to many other evolutionary changes, at a physiological level, such as those you suggest in your very interesting comment.