The nonmammalian-mammalian transition through neurohypophysial peptides

Peptide~. Vol. 6, Suppl. 3, pp. 30%314. 1985. ' Ankho International Inc. Printed in the U.S.A.

0196-9781/85 $3.00 + .00

The Nonmammalian-Mammalian Transition Through Neurohypophysial Peptides ROGER ACHER

Laboratoo' of Biological Chemistry, Universit~ Pierre et Marie Curie, 96 Boulevard Raspail F-75006 Paris, France

ACHER. R. The mmmammalian-nuonmalian transition through neurohypophysial peptides. PEPTIDES 6: Suppl. 3, 30%314, 1985.--Neurohypophysial hormones are particularly proper evolutionary tracers. Whereas Eutherian mammals have oxytocin and virtually always arginine vasopressin, nonmammalian tetrapods possess instead mesotocin and vasotocin. The transitions of mesotocin-oxytocin and vasotocin-vasopressin involved in the passage of reptiles-mammals seem to have occurred independently. Australian marsupials are endowed with mesotocin but American marsupials have either oxytocin (South-American opossums) or both oxytocin and mesotocin (North-American opossum). These results suggest that Australian Metatherians have preserved reptilian mesotocin and used it for milk-ejecting function whereas the change mesotocin-oxytocin appeared only in the American line. All marsupials have either arginine vasopressin or lysipressin and phenypressin (Australians) or lysipressin and arginine vasopressin (Americans). It is assumed that the change of vasotocin into arginine vasopressin occurred very early, perhaps in mammal-like reptiles, and duplication of the gene with subsequent mutations has led to the presence of two vasopressin-like peptides in most Metatherians. Neurohypophysial hormones

Oxytocin

Arginine vasopressin

P U R I F I C A T I O N O F T H E PEPTIDES

Mesotocin

Vasotocin

ders; the living Metatheria form a single order, the Marsupiala.

Progressive miniaturization in purification technology and sequencing analysis has now made possible the chemical identification of neurohypophysial peptides from any vertebrate species. The use of counter-current distribution in the fifties allowed isolation and characterization of neurohypophysial peptides from large mammals whereas the procedure of complexation between hormones and neurophysins opened the way, in the sixties, to neurohypophysial peptides from small but abundant vertebrates. Paper chromatoelectrophoresis made accessible rare hinge species, such as lungfishes or egg-laying mammals, in the seventies, and high pressure liquid chromatography (HPLC) now permits study on individual glands of small experimental animals such as rats. The amount of material necessary for the nonapeptide characterization has decreased from 100,000 nmol to 1 nmol within thirty years (Fig. 1). More than 50 vertebrate species have been examined to date and 10 distinct neurohypophysial peptides have been identified [i]. The transition between reptiles and mammals occurred with a dramatic change in the nature of neurohypophysial hormones.

Eutherian Hormones Nine eutherian species have been studied from six orders: Primates, Artiodactyla, Perissodactyla, Cetacea, Rodentia, and Lagomorpha: all were found to possess essentially the same active principles. Oxytocin and arginine vasopressin have been characterized in man, ox, sheep, horse, whale, rat, guinea pig and rabbit [1]. In the domestic pig, lysine vasopressin ([LysS]-vasopressin) takes the place of arginine vasopressin, differing from the latter in the substitution of the lysine residue for that of arginine (Table I).

Metatherian Hormones Recent investigations on marsupials have revealed that metatherians may have neurohypophysial hormones different from those of Eutherians. Living metatherians are confined in particular continents, namely North and South America on one hand, Australasia on the other. Whereas American marsupials belong almost exclusively to a single family, Didelphidae, Australasia species are classified in 6 to 13 families according to the different authors [i 1]. Australasia marsupials. Up to now two families have been investigated, Macropodidae and Phahmgeridae. The common and striking feature is the presence of mesotocin instead of oxytocin found in eutherians [2,9]. Because mesotocin has previously been identified in all the nonmammalian tetrapods, it may be regarded as a relatively " o l d " or "primitive" hormone. The interesting fact, however, is the

C H A R A C T E R I Z A T I O N O F THE PEPTIDES MAMMALIANHORMONES Rothschild [13] recognizes two subclasses in Mammalia: the Prototheria, represented by a few rare species, and the Theria, comprising the Metatheria and the Eutheria. The Eutheria represent 98% of the mammals, classified in 17 or-

309

ACHER

310

TABLE 1 STRUCTURESOF THE NEUROHYPOPHYSIALHORMONES Oxytocin-Like Peptides I

2

3

4

5

6

7

Vasopressin-Like Peptides

8

9

1

2

3

4

5

6

7

8

9

Cys-Tyr-Ile-GIn-Asn-Cys-Pro-Leu-Gly(NH.,)

Cys-Tyr-Phe-GIn-Asn-Cys-Pro-Arg-Gly(NH.,.)

Oxytocin (Prototherian, metatherian (American) and eutherian mammals)

Arginine vasopressin (Prototherian. metatherian and eutherian mammals)

Cys-Tyr-Ile-Gln-Asn-Cys-Pro41e-Gly(NH.,.)

Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly(NH.~)

Mesotocin (Australian marsupials, birds, reptiles, amphibians, lungfishes)

Lysine vasopressin (Eutherians: pig: metatherians)

Cys-Tyr-lle-Ser-Asn-Cys-Pro-lle-Gly(NH.,_)

Cys-Phe-Phe-GIn-Asn-Cys-Pro-Arg-Gly(NH.,)

lsotocin (Bony fishes)

Phenypressin (Metatherians: macropodids)

Cys-Tyr-Ile-Ser-Asn-Cys-Pro-GIn-Gly(NH._,)

Cys-Tyr-lh,-Gln-Asn-Cys-Pro-Arg-Gly(NH,)

Glumitocin (Cartilaginous fishes: rays)

Vasotocin (Nonmammalian vertebrates)

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Val-Gly(NH.,.) Valitocin (Cartilaginous fishes: sharks)

Cys-Tyr-Ile-Asn-Asn-Cys-Pro-Leu-Gly(NH.,.) Aspargtocin (Cartilaginous fishes: sharks)

PURIFICATION OF NEUROHYPOPHYSIAL HORMONES ~,oum Oe~CETONE PERIOD

1950-1958 1958-1969

1969-t982

1982-198

S P E C I E 5 POSTERIOR PITUITARY

POeOER~

Ox

1 O0

NUMBEROF GL~'M[~

1.00 0

Chicken

1

1.500

Frog

1

10.000

Iguana

0.130 tentie-~

Echidna

0060

Kanqaroo

0.040

8

Marsupialrot

0.007

12

100 19

FIG. 1. The four periods correspond essentially to the use of counter-current distribution, neurophysin complexation, chromatoelectrophoresis and high pressure liquid chromatography.

emergence of mesotocin receptors in the new mammary gland so that, aside from the former action on the oviduct or the uterus, mesotocin becomes involved in lactation as a galactagogic hormone. Mesotocin ([IleS]-oxytocin). The peptide mesotocin was first isolated from the posterior hypophysis of the frog Rana esculenta by the protein-complex technique [1]. Isolation was later possible with a few hundred glands through chromatoelectrophoresis and with a few glands through HPLC in the case of marsupials. The amino acid sequence consists of nine resides similar to those of oxytocin, except

that leucine in position 8 is replaced by an isoleucine residue (Table 1). Another unexpected finding in Metatherians is the duplication of the pressor peptide in Macropodidae [2] whereas the single species of Phalangeridae examined, the brushtailed possum, has only arginine vasopressin as eutherians [9]. In six species of Macropodidae, including kangaroos and wallabys, two pressor peptides have been characterized: lysipressin (lysine vasopressin), previously found in pigs. and phenypressin. Because the two hormones have been detected in all the individuals examined, it is likely that they are products of two separate genes rather than two products of a single gene (heterozygosity). Phenypressin ([Phe2]-vasopressin). Phenypressin is a peptide composed, as the other known neurohypophysial hormones, of nine amino acid residues with a disulfide bridge 1-6. Amino acid analysis reveals two phenylalanine residues per mole and a lack of tyrosine; amino acid sequence is similar to that of arginine vasopressin, with a replacement of tyrosine in position 2 by phenylalanine [2]. (Table 1) American marsupials. The three species investigated to date belong to the family Didelphidae. The North American opossum, Didelphis virginiana, has four hormones: oxytocin, mesotocin, lysine vasopressin and arginine vasopressin [3]. On the other hand, two South American didelphids. Didelphis marsupialis and Philander opossum, have three peptides: oxytocin, lysine vasopressin and arginine vasopressin [4]. It is not yet known whether the dimorphism of the vasopressin-like peptides results from a gene duplication or from heterozygosity. Similarly, it is not determined whether the dual presence of oxytocin and mesotocin in the North American opossum in due to a gene duplication or to two alleles of a single gene. It is puzzling to find oxytocin in American marsupials and mesotocin in Australian metatherians.

N EUROPEPTIDE TRACERS IN REPTILE-MAMMAL TRANSITION

Prototheriau Hormones The two living egg-laying mammals, namely echidna and platypus, have been investigated [1,5]. Despite the fact these species are supposed to have diverged very early from the mammalian stem, some 200 million years ago [12], they have the same neurohypophysial peptides as placental mammals, namely oxytocin and arginine vasopressin.

311

MAMMALS

0

0

MAMMALS

0~0

AV AV AV

O

0~ fO~M

LV AV AV

AV

ilrllJl tl -

~

M

MAMMALS

M ~

0

LV AV

tV PP AV

AV

•

~

o

~

BIRDS REPTItES

AT

AT

AT

f Idf

t,Om 70m HORMONES OF THE NONMAMMALIAN TETRAPODS

Isolation and characterization of the hormones in birds, reptiles, and amphibians showed that these vertebrates possess identical hormones, and thus, from endocrine point of view, the three classes show a surprisingly close relationship. In the ten species studied, two peptides, mesotocin and vasotocin, were found. Since vasotocin appears to play the same antidiuretic role as do vasopressins in the mammals, it is reasonable to assume that it has given rise to mammalian vasopressins and similarly that mesotocin preceded oxytocin. Nevertheless, although the function of oxytocin in mammals is associated with reproduction because of its uterotonic and milk-ejecting properties, that of mesotocin in birds, reptiles and amphibians remains discussed. Vasotocin ([lle3]-vasopressin). Vasotocin was first isolated from chicken posterior hypophyses by means of the protein complex technique [1]. Subsequently the method was successfully used for the frog Rana esculenta and the toad Bufo bufo. Later vasotocin has been purified by the paper microtechnique from glands of goose, turkey, viper, cobra, elaphe, iguana and leopard frog [1]. The amino acid sequence of vasotocin consists of nine residues similar to those of arginine vasopressin except that phenylalanine does not occur in position 3, being replaced by an isoleucine residue tTable I). A clear-cut difference regarding neurohypophysial hormones seems therefore to exist between the mammals and the other tetrapods: vasotocin and mesotocin of the latter are replaced by a vasopressin and oxytocin in the former, with maintenance of mesotocin in Australian and North American marsupial species. HORMONESOF THE BONYFISHES Bony fishes are the most diversified of the seven vertebrate classes, being represented by about 20,000 species, approximately half of all living vertebrates. Rothschild [13] distinguishes three subclasses: Paleopterygii (Chondrostei and Cladistia), Neopterygii (Teleostei and Holostei) containing nearly all living bony fishes, and Crossopterygii (Dipnoi and Coelacanthini) related to the ancestral amphibians. Study of a few species from each of these three sub-classes shows that whereas all possess vasotocin, the first two groups have characteristic oxytocin-like hormones. The Dipnoi of the third group do in fact possess the nonmammalian tetrapod hormones.

Crossopter3"gians The crossopterygian group includes the coelacanths and the lungfishes [13]. Mesotocin has been isolated and characterized in two protopterans, Protopterus annectens and Protopterus dolloi, found in West and South Africa, respectively [1]. A second hormone was characterized only pharmacologically because of the lack of material, and shown to be vasotocin. Certain pharmacological data indicate the

100m

v2B0m

FIG. 2. Neurohypophysial hormones and species evolution according to paleontological data. Letters indicate hormones identified in modern representatives of groups: O, oxytocin; M, mesotocin; AV, arginine vasopressin; LV, lysine vasopressin; PP, phenypressin; AT, vasotocin. Numbers give time in millions of years (m) since divergence.

presence of mesotocin and vasotocin in the third species of African protopteran, P. aethiopicus [8,16]. These hormones have also been pharmacologically recognized in the Australian lungfish Neoceratodus forsteri [8], and thus it is evident that Dipnoi and Amphibia possess the same active principles that were present in their common ancestors.

Neopterygians Hormones have been isolated from eight species belonging to the families Gadidae, Scombridae, Cyprinidae, and Salmonidae. The same peptides, isotocin and vasotocin, were identified in all cases. Isotocin ([Ser4,lleS]-oxytocin). The peptide isotocin was first purified from entire hypophyses of pollack, whiting pout and hake, and subsequently of cod, tunny fish, mackerel, carp [1] and salmon [19]. Its amino acid sequence indicates that it contains nine residues, as does oxytocin, but the glutamine and leucine residues in positions 4 and 8 are replaced by serine and isoleucine, respectively (Table 1).

Palaeopte~.'gians The subclass Palaeopterygia contains two orders grouped together because of their archaic characters: the Chondrostei (sturgeons) and the Cladistia (polypterans). In the Chondrostei, vasotocin has been chemically characterized [1]. A second hormone, different from those found in other fishes, has not yet been identified. The Cladistia are represented by eight species of polypterans, all native to Africa. In Polypterus bichir, both isotocin and vasotocin have been isolated and analyzed. In another species. P. senegalus, the two hormones have been pharmacologically recognized [ 16]. The polypterans, generally considered to be very primitive bony fishes, possess, therefore, the same hormones as the neopterygians, and these peptides probably already existed in their common ancestors.

312

ACHER THE VASOPI:ESSIN~L-NE~YSIN

GEE

MSEL" i~" G L~'(:O -~ L ~ .

78-93 In,ton t EXON

A

(lO00bpl

(l.Ore~kJesJ

EXON B [68re$.~,es}

INTERGENE VARIABLE EXON

INTERGENE CONSTANT EXON

Intr-on II (227bp1

I~.~tD E.~ ~

EXON

C

[56 r,~du, s]

INTRAGENE VARIABLE EXON

T H E OXYTOCIN/VLDV-NEUROPHYSIN GENE

5"--

t$ ['\ \ IGNAL[OXY~ \ ' \~x~X('SJ] VLDV- L - - . - . ~ | \ \ \ \ \\ \ "~ll/~l 1.9 j

l

VL OV n eu e ° Ph Y~"n

~0.77

FIG. 3. Rat vasopressin and oxytocin genes and their exons Iboxes). AVP or OXY. nucl¢otide sequences encoding arginine vasopressin or oxytocin, respectivel.~. MSEL- or VLDV-I-9. 1(~-77.78-93, nucleotide sequences encoding the corresponding residues of MSEL- or VLDV-neurophysins. respectively. Glycopeptide. nucleotide sequence encoding a 39-residue gl,,copeptide. R: codon for an arginine residue ¢Redrawn fiom [10]L

HORMONES OF THE CARTILAGINOUS FISHES

HORMONES OF THE CYCI.OSTOMES

Chondrichthyes. a heterogeneous class, contains the subclasses Selachii (sharks and rays) and Holocephali (chimaeras). the latter being considered by some authors as a separate class [13]. The neurohypophysial hormones of the cartilaginous fishes differ from those in other classes and also show considerable variation within their class. Although sharks, rays. and chimaeras all possess vasotocin, they show individual differences as regards the second hormone.

Identification of the active principles in Cyclostomes i, of great interest, since the Cyclostomata is the oldest vertebrate class, having existed for about 450 million years. It is difficult to work with this group, however, because a few living species exist and, judging by the pharmacological activities of their hypophysial powders, the bios} nthesis of the hormones is very low. Studies so far undertaken on the lamprey and the hagfish have only revealed the presence of arginine ~asotocin. the peptide being identified by its electrophoretic and chromatographic behavior, and also by its biological properties [7.15r.

Hypatremata (Rays) Isolation and amino acid analysis of the active principles have only been carried out extensively in rays. in which glumitocin has been identified in four species: Raia clavatu. R. bati,L R. naevtts and R. fulhmica [ I]. Ghunitocin ([SerLGIn~]-o.U'tocin). The amino acid sequence ofglumitocin consists of nine residues as in oxytocin except for the replacement of glutamine-4 and leucine-8 by serine and glutamine, respectively (Table 1).

Ph'urotrcmata (SharL~) Two particular oxytocin-like hormones, valitocin and aspargtocin, have been characterized in the spiny dogfish. Squahts aca/tthias [I ]. On the other hand, arginine vasotocin in small amount has been recognized pharmacologically in this species. Valitocin and aspargtocin were isolated from acetone powders prepared from many dogfish pituitaries and it is not known whether the two peptides exist in the same individual. Valito(in ([Val"]-o.vytocin). The amino acid sequence of valitocin is similar to that of oxytocin except that leucine in position 8 is replaced by valine (Table I). Aspar~,to('in ([Astt4]-oATtocin). The amino acid sequence of aspargtocin is similar to that of oxytocin except that the glutaminyl residue in position 4 is replaced by an asparaginyl residue (Table I).

EVOLUTION O F THE PEPTIDES Up to now, six oxytocin-like hormone,, and four vasopressin-like hormones have been identified. All the~e peptides have nine amino acid residues, five of which are invariant. They can be arranged in two lineages corresponding to the oxytocin-like and vasopressin-like hormones. The fi'equent duplication of the vasopressin-like gene in mar,upials suggests the existence in this group o f a multigene f:mlil.,. for pressor peptides. On the other hand the co-presence of mesotocin and oxytocin in the North American opos,um may also be due to a gene duplication. T H E L I N E A G E ¢)F ( ) X Y T O C I N - L I K E H O R M O N E S

In the oxytocin-like family, substitutions occur onl~ in positions 4 and/or 8 (Table I), In position 4. a polar residue. namely glutamine, serine, or asparagine, is airways pre,,enl. In position 8. a nonpolar residue such as leucinc, isolcucinc or valine is almost alwa}s present with the exception of gltttamine in glumitocin. A possible evolution of an ance,tral "'protolocin'" gene could have given isotocin in bony fishes. glumitocin in rays and, after duplication, valitocin arid ;tspargtocin in sharks. Mesotocin is supposed to be deri,.cd fiom bony fish isotocin. It persisted in lungfishes, amphi-

N E U R O P E P T I D E T R A C E R S IN R E P T I L E - M A M M A L TRANSITION bians, reptiles, birds, and Australian marsupials, but was apparently replaced by oxytocin in Prototherians, South American marsupials, and placental mammals (Fig. 2). THE LINEAGE OF VASOPRESSIN-LIKEHORMONES In the vasopressin-like family, substitutions occur only in positions 2, 3 or 8 (Table 1). Vasotocin has been found in all the vertebrates except in adult mammals (Fig. 2). Because of the presence of arginine vasopressin in the primitive prototherians, it may be assumed that the change (substitution o f isoleucine by phenylalanine in position 3) occurred early in mammals or even in mammal-like reptiles. Arginine vasopressin remained during eutherian evolution with rare change into lysine vasopressin (substitution of arginine by lysine in position 8) in the pig [6] and Peru mouse [17]. In the course of metatherian evolution, arginine vasopressin remained alone in the Australian family Phalangeridae. In another Australian family, the Macropodidae, a duplication of the structural gene followed by point mutation led to [LysS]-vasopressin (lysine vasopressin or lysipressin) and [Phe2]-vasopressin (phenypressin). In South and North American opossums (Didelphidae), an apparent duplication also occurred but the two pressor hormones are arginine vasopressin and lysipressin (Fig. 2). The three vasopressin-like hormones of mammals have a strong antidiuretic activity on the rat kidney but have lost the secondary activities on the uterus and mammary gland displayed by vasotocin. THE NONMAMMALIAN-MAMMALIAN TRANSITION Because on one hand mesotocin and vasotocin have been found in nonmammalian tetrapods, particularly in reptiles, and oxytocin and arginine vasopressin in placental mammals, it has been assumed that oxytocin derived from mesotocin by a point mutation in the gene, and vasopressin derived from vasotocin in the same way [1]. Oxytocin and vasopressin genes o f rat [10] and ox [14] have been recently characterized and shown to be built in a similar way by three exons separated by two introns (Fig. 3). Each hormone moiety is encoded by the first exon that includes the coding

313

sequence for the first 9 residues of a neurophysin. Vasopressin-associated neurophysin is termed MSELneurophysin according to the nature of amino acids in positions 2, 3, 6 and 7 and oxytocin-associated neurophysin is called VLDV-neurophysin for the same reason [1]. Each gene is expressed into a protein precursor that is processed, in the synthesizing neuron, into an active nonapeptide, a neurophysin and a glycopeptide in the case of vasopressin precursor. It can be supposed that oxytocin and vasopressin genes result from a primordial gene duplication that occurred before the emergence of fishes. Although single-gene evolutions could explain the two lineages of neurohypophysial hormones, the co-presence of mesotocin and oxytocin in the North American opossum suggests a possible two-gene evolutionary mechanism. A duplication o f the mesotocin gene, perhaps in some mammal-like reptiles, could have led, after mutation in one copy, to the simultaneous presence o f mesotocin and oxytocin. Another mutation could have made the mesotocin gene silent (pseudogene) and only oxytocin gene would have remained expressed in South American marsupials. This interpretation needs to find two distinct oxytocin-like genes in the North American opossum genome. In the same way, the passage from vasotocin to vasopressin could be explained either by a point mutation in a single gene or by a two-gene mechanism. The co-presence of vasotocin and vasopressin has yet not been found in any mammalian species. However in sheep and seal foetuses at mid-gestation this co-presence has been noted, with disappearance of vasotocin at the birth [18]. If this finding is confirmed, it would suggest the existence of a vasotocinvasopressin multigene family with successive expressions during development, as found for instance for hemoglobins. Additional duplications followed by point mutations of the vasopressin gene seem to have occurred in several marsupial families. It is of interest to find this putative duplication in both Australian and American marsupials. The common origin of all present-day metatherians with migrations along the North America, South America, Antarctica, Australia garland, suggested by paleontological data [ 12], might be confirmed by molecular biology.

REFERENCES

I. Acher, R. Molecular evolution of biologically active polypeptides. Proc R Soc Lond [Biol] 210: 21-43, 1980. 2. Chauvet, M. T.. D. Hurpet, J. Chauvet and R. Acher. Identification of mesotocin, lysine vasopressin and phenypressin in the Eastern gray kangaroo (Macropus giganteus). Gen Comp Endocrimd 49: 63-72. 1983. 3. Chauvet, J., D. Hurpet, G. Michel, M. T. Chauvet and R. Acher. Two multigene families for marsupial neurohypophysial hormones? Identification of oxytocin, mesotocin, lysipressin and arginine vasopressin in the North American opossum (Didelphis virginiana). Biochem Biophys Res Commun 123: 306311, 1984. 4. Chauvet, J., D. Hurpet, T. Colne, G. Michel, M. T. Chauvet and R. Acher. Neurohypophyseal hormones as evolutionary tracers: Identification of oxytocin, lysine vasopressin, and arginine vasopressin in two South American opossums (Didelphis marsupialis and Philander opossum). Gen Comp Endoc'rinol 57: 320-328, 1985.

5. Chauvet, J., D. Hurpet. G. Michel. M. T. Chauvet, F. N. Carrick and R. Acher. The neurohypophysial hormones of the egglaying mammals: Identification of arginine vasopressin in the platypus (Ornithorhynt'hus anatinus). Biochem Biophys Res Commun 127: 277-282, 1985. 6. Du Vigneaud, V. Hormones of the posterior pituitary glands: oxytocin and vasopressin. Harvey Lectures. Ser. 50, 1-26, 1954-1955. 7. Follett, B. K. and H. Heller. The neurohypophysial hormones of bony fishes and cyclostomes. J Physiol (Lond) 172: 74--91, 1964. 8. Follett, B. K. and H. Heller. The neurohypophysial hormones of lungfishes and amphibians. J Physiol (Lond) 172: 92-106, 1964. 9. Hurpet, D., M. T. Chauvet, J. Chauvet and R. Acher. Marsupial hypothalamo-neurohypophysial hormones. The brush-tailed possum tTrichosurus vtdpecuht) active peptides, lnt J Pept Protein Res 19: 366--371, 1982.

314 10. Ivell, R. and D. Richter. Structure and comparison of the oxytocin and vasopressin genes from rat. Proc Natl Acad Sci USA 81: 2006--2010, 1984. I 1. Kirsch. J. A. and J. H Calaby. The species of living marsupials: an annotated list. In: The Biology of Marsupials, edited by B. Stonehouse and D. Gilmore. Baltimore: University Park Press, 1977, pp. 10--26. 12. Romer, A. S. Vertebrate Paleontology. Chicago: Univ. Chicago Press, 1966. 13. Rothschild. N. M. V. A ClassiJ~cation of Living Animals, 2nd edition. London: Longman, 1965. 14. Ruppert, S., G. Scherer and G. Schutz. Recent gene conversion involving bovine vasopressin and oxytocin precursor genes suggested by nucleotide sequence. Nature 308: 554-557, 1984.

ACHER 15. Sawyer, W. H., R. A. Munsick and H. B. Van Dyke. Pharmacological characteristics of the active principles in neurohypophysial extracts from several species of fishes. En&~crinology 67: 136-138, 1961. 16. Sawyer, W. H. The active neurohypophysial principles of two primitive bony fishes, the bichir (Polypterus senegalis) and the African lungfish (Protopterus aethiopicus). J Endocrinol 44: 421--435, 1969. 17. Stewart, A. D. Genetic variation in the neurohypophysial hormones of the mouse (Mtts mtts¢'tthts). J Endocrinol 41: 1%20. 1968. 18. Vizsolyi, E. and A. M. Merks. New neurohypophysial principle in foetal mammals. Nature 223:116%1170, 1969. 19. Wilson, N. and M. Smith. Isolation and amino acid sequence of neurohypophyseal hormones of Pacific chinook salmon (Oncorhynchus tschawyts('ha). Gen Comp Endoc'rinol 13: 412-424, 1969.