The Influence of Anterior-Pituitary and Testicular Hormones on the Sexual Maturation of Lampreys

It is known that the pituitary gland of the higher vertebrates regulates many of those physiological activities by which animals attain sexual maturity. Less information, however, is available about the regulation of the sexual development of the lower vertebrates, especially the fishes. To judge from a few reports, the gametogenesis and the formation of the secondary sexual characters of certain teleosts may be influenced by mammalian-pituitary preparations (see Schreiber, 1937), but the capacity to respond to these preparations appears to be confined to a few species, and is not a general feature of the group. No experiments have yet been performed which indicate that the sexual development of elasmobranchs may be affected by mammalian hormones.

Certain observations, however, suggest that the development of cyclostomes may be influenced by mammalian-pituitary preparations. Damas (1933) and Young & Bellerby (1935), have recorded a swelling of the cloacal labia in immature lampreys which had been injected with pituitary preparations.

Comparable swellings do not appear in the adult until maturity. Damas (1933) and Calvet (1932) have further claimed that the maturation of the lamprey’s gonad may be accelerated by mammalian-pituitary preparations. It appears, therefore, that a relationship between the pituitary and the regulation of maturity may have appeared early in the evolution of the vertebrates.

In mammals, the secretions of the pituitary stimulate the gonads to secrete hormones, which act on the body tissues to produce secondary sexual characters. It has not been previously investigated whether mammalian gonadial hormones will produce changes in the cloacal labia of cyclostomes, or whether the response of these labia is specific to pituitary preparations. The present paper describes observations made on the maturation of the gonads, and the development of the accessory body modifications in river lampreys living under natural conditions, and in individuals which had received injections of anterior-pituitary preparations, testicular or ovarian hormones.

Immature adult river lampreys of the species Lampetra fluviatilis were obtained from fisheries on the river Severn, and kept in outdoor tanks with a constant water circulation at the Department of Zoology, Oxford. These lampreys enter the river Severn from the sea during the autumn months, and spawn in the following spring. While in the river, they do not feed, but depend on the food reserves which they have accumulated during their life in the sea. In the spring their gonads mature, and external secondary sexual characters make their appearance. From the available data it would seem that, in the Severn, the river lamprey spawns in April.

A number of these adult lampreys were injected with pituitary preparations and gonadial hormones in January and February 1937. A later series of experiments was made on ammocoete larvae at the Stazione Zoologica, Naples, Italy, during 1938. The ammocoete larvae were obtained from the river Sarno. They were kept in indoor tanks at the Stazione Zoologica and were injected with pituitary preparations and testicular hormones.

The injections during the preliminary tests were given intraperitoneally, but it was found later that intramuscular injections were more effective, and this latter method was adopted in subsequent experiments on adults and larvae. Although less volume of fluid can be injected into the muscles than into the coelom, there is a greater chance that the injected fluid will be absorbed by the body tissues. It was noted in the first experiments that any violent movements of the animals tended to expel the injected fluids from the coelom.

The pituitary preparations used in the experiments were kindly supplied by Dr A. S. Parkes, F.R.S., and the British Medical Research Council. The pituitary preparations were extracts of the mammalian anterior-pituitary gland. Extract A.P. 6B was prepared from mare’s pituitary gland. It was an alcohol precipitate from an N/20 NaOH extraction of acetone-desiccated anterior-lobe pituitary tissue. Extract A.P. 15 B was an alcohol precipitate from a 50 % pyridine extract of acetone-desiccated ox anterior-pituitary gland. These extracts are known to have a gonadotropic action in the mammal. A purified thyreotropic extract (A.P. 32D) was also used. For the gonadial hormones I am indebted to Dr S. Zuckermann of the Department of Anatomy, Oxford. The male “sex-hormone” was a commercial preparation by Ciba Ltd. of testosterone propionate.

The functional kidney of cyclostomes is a mesonephros, whose coelomostomes are never open in Lampetra. In the ammocoete larvae the paired mesonephric ducts join the rectum and the resultant urino-rectal sinus opens to the exterior by a small median ventral opening, the cloaca,¹ which is bounded by two lateral labia. These cloacal labia are folds of the dermis, supported by connective tissue. At no stage in the larval life is there any open communication from the coelom to the urino-rectal sinus (Text-fig. 1).

The condition of the ducts of the adult differ from that in the ammocoete in that the rectum does not join the urinary ducts, but opens separately into the cloaca. The paired mesonephric ducts fuse to form a urinary sinus, opening to the exterior independently of the rectum by a urinary papilla for the emission of genital products.

At maturity the genital products are shed freely into the coelom, whence they escape to the exterior through the urino-genital papilla. In the immature adult there is no open communication from the coelom to the urinary sinus, but, at complete maturity, just posteriorly to the place where the two mesonephric ducts join, the urinary sinus develops a pore, on each side, leading into the coelom (Text-fig. 2). It is through these pores that the genital products escape to the exterior. Shortly before spawning the cloacal labia become enlarged on account of a vasodilatation of the blood vessels supplying the cloacal region. In the male lamprey this excess of blood results in the erection of the urino-genital papilla. The sperms, therefore, are not shed into the cloaca, but are directly ejected to the exterior. The cloacal region of the female lamprey also becomes swollen and hyperaemic before spawning.

These external and internal changes in the cloaca occur only a few days before spawning, and aid the deposition of sperms and eggs. They are, however, preceded by the appearance of differences between the two sexes. A month before spawning the dorsal and caudal fins enlarge, and become united by a thickening at their base, developing a swelling which is much more conspicuous in the female than the male. A small ventral fin develops behind the cloaca of the female lamprey ; this fin does not develop in the male. Since in other respects the external features of male and female lampreys are similar, these differences may help in the recognition of the opposite sex at the time of spawning.

The injection of testosterone, oestrone, and anterior-pituitary preparations had a marked effect on the cloacal region of L. fluviatilis. The cloacal labia of both males and females became very swollen and hyperaemic as a result of these injections. Young & Bellerby (1935) have reported similar effects in the closely-allied species L. planeri after the injection of a mammalian-pituitary preparation.

Table I summarizes the results obtained by injecting the adult males with various preparations. Testosterone and oestrone produced a more rapid response of the cloaca than did the pituitary preparation, and this response was more marked in the animals approaching sexual maturity. Injections of boiled pituitary extract, aqueous oestrin, or water, had no effect on the cloacal region, although a posteriorpituitary preparation (pitressin), had a slight effect, producing a hyperaemia, but no swelling of the cloacal labia. Intramuscular injections were more effective than intraperitoneal administrations, thus indicating that an increased pressure in the coelom was not a contributory factor to the cloacal response.

The injection experiments were commenced in January 1938, and continued until the end of February. They were then stopped, as modifications in the fins and urino-genital papilla were observed in many of the uninjected controls during the first weeks in March. The fin characters did not appear at an earlier date in the lampreys which had been injected with pituitary or gonadial hormones, even though modifications of the cloacal labia appeared in these animals. Damas (1933) has reported that the appearance of fin modifications can be accelerated by the injection of mammalian pituitary preparations. In the present series, occasional animals showed these secondary sexual characters in February, but such early fin modifications were found in both experimental animals and members of the normal population.

Table I shows the cloacal region of adult lampreys to be sensitive to the injection of gonadial hormones and of pituitary preparations. Whereas the swellings were generally more marked and rapid in the adult animals injected with gonadial hormones than those injected with pituitary preparations, subsequent experiments showed that in ammocoetes the reverse is the case. Pituitary preparations were the more effective agents for inducing comparable changes in the cloacae of ammocoetes (Table II), a testicular hormone producing only occasional and slight effects.

Testosterone propionate was not usually effective for inducing the formation of cloacal swellings in ammocoetes, either by intramuscular or by intraperitoneal injections. The cloacae of two animals in a batch of ten ammocoetes were slightly swollen after an injection of 1 mg. of testosterone propionate. (A similar dose produced a marked effect in all the adults of L.fluviatilis.) In a later experiment, an injection of 1 mg. of a mammalian pituitary preparation (A.P. 15 B) was sufficient to produce swollen and hyperaemic cloacal labia in sixteen out of twenty injected animals. This response was very rapid, in many cases occurring 24 hr. after injection. Pituitary preparation A.P. 6B was also tested on ammocoete larvae and found to produce comparable cloacal changes.

It is of interest to note that the frequency of the cloacal response to pituitary preparations varies with the size, and therefore, the age of the larvae. If the larvae are less than 110 mm. in length, their cloacal labia do not respond to doses of the hormone which will have a marked effect on the cloacae of larger animals.

No cloacal response was obtained in the ammocoete to the injection of water or to a pituitary extract which had been de-activated by boiling. A posterior-pituitary extract (pitressin) produced slight hyperaemia but no swelling of the cloacal labia. From these experiments it would appear that the response of the larval cloaca is produced mainly by pituitary preparations, the effect of a testicular hormone being slight or absent. No cloacal response was obtained by the injection of a preparation which contained only the thyreotropic principle of the mammalian pituitary, which indicates that the gonadotropic principle was the fraction responsible for the cloacal swellings of those ammocoete larvae which were injected with whole anterior-pituitary preparations. However, it is possible that the hormone present in the pituitary preparations which affects the cloacae of lampreys is not one of the recognized mammalian hormones. Shapiro & Zwarenstein (1937) found that the amphibian cloaca became swollen as a response to some hormone produced by the amphibian ovary when it is caused to ovulate, and suggested that this hormone was not the same as any of the known gonadial hormones.

A histological examination of the cloacae in animals which had been injected with pituitary preparations or gonadial hormones, showed various features which were not present in the uninjected controls. The swelling of the cloacal labia was seen to be the result of an infusion of blood to this region: the small blood vessels which supply the connective tissues surrounding the urinary sinus and cloacal labia became filled with blood. No hypertrophy of the tissues forming the cloacal labia was apparent, but the excess of blood in the cloacal labia would seem sufficient to account for the swelling and hyperaemia of these tissues after injection.

In the male the urino-genital papilla was extended as a result of the quantity of blood in the blood vessels which lie between the two layers of dermis in this organ. In the female the cloacal labia were also swollen, but there was no erection of the relatively small urino-genital papilla found in this sex.

In the immature adult, the posterior ends of the coelom closely approach the mesonephric ducts on both sides. They are, however, separated from these by the epithelium lining the mesonephric ducts, and by a small quantity of connective tissue. In animals injected with A.P. 6B, testosterone propionate, or oestrone, this separation disappeared, due to a degeneration of some of the cells which form the walls of the mesonephric ducts. This condition may be seen in Pl. II, where, although an open communication from the coelom to the mesonephric ducts has not yet completely formed, yet the nuclei of many of the cells in the mesonephric wall near the coelom show unmistakable signs of degeneration. This formation of a pore from the coelom to the mesonephric ducts on both sides, was found in both male and female animals, which had been injected with pituitary preparations’ or male or female gonadial hormones. It was found in animals which were killed as early as 29 January and was yet more marked in animals which were killed at a later date. In the normal population of uninjected controls the separation between the coelom and mesonephric ducts persisted until March, although in many cases there were free sperms or eggs present in the coelom before any pore had formed.

This accelerating effect of pituitary preparations and gonadial hormones on the development of the cloacal modifications which normally only appear at maturity, indicates that the normal appearance of these characters in the lamprey is influenced by the pituitary or gonads, but it is not clear which of these two glands is responsible for the effect.

Normally no open communication from the coelom to the cloaca is ever seen in ammocoete larvae. However, it was found that pores from the coelom to the urino-rectal sinus very readily developed as a result of the injection of pituitary preparations. A disappearance of the tissues separating the coelom from the urino-rectal sinus, resulted in the formation of short ducts from the coelom to this sinus. Pl. I shows the gradual formation of these “ducts” in ammocoetes (115 nun. in length) which had received doses of various concentrations of preparation A.P. 15 B. In the normal ammocoete the coelom is separated from the urino-rectal sinus by the rectal wall, the coelomic epithelium, and a considerable amount of connective tissue (Pl. I, fig. 1). It may be noted that the coelomic epithelium is particularly thick at the points nearest to the urino-rectal sinus, where it forms “pads”, probably to resist the pressure exerted by the faeces in the rectum. Blood vessels occupy a considerable space in the connective tissues surrounding the rectum, but in such normal ammocoetes they are small, and not distended by blood.

An intramuscular injection of $12$ mg. of A.P. 15 B produced marked internal changes in the cloacal region within 48 hr. (Pl. I, figs. 2, 3). The blood vessels surrounding the urino-rectal sinus became distended with blood, the rectal wall thickened at the point nearest to the coelom, and the pads of coelomic epithelium disappeared. Later, the cells of the rectal wall showed signs of degeneration at the points where the wall had thickened, thus forming two pores in the wall of the urino-rectal sinus.

These modifications were yet further accentuated in animals which had received injections of 1 mg. of A.P. 15 B (Pl. I, fig. 4). The blood spaces round the rectum were enormously distended and filled with blood, the coelomic epithelium was no longer present, and, in many animals, complete “ducts” existed from the coelom to the urino-rectal sinus. In the individual whose cloacal region is figured in Pl. I, fig. 4 and Pl. II, fig. 1, a thin membrane still separates the coelom from the urino-rectal sinus. The presence of this membrane shows that the degeneration of the cells in the wall of the urino-rectal sinus is not the result of a mechanical pressure exerted upon them by an increased quantity of fluid in the coelom or urino-rectal sinus.

Therefore, it seems clear that pituitary preparations have an effect on the cells in the wall of the urino-rectal sinus and on certain cells of the coelomic epithelium in ammocoetes and that the response of these cells seems specific to pituitary preparations, since neither testosterone (2 mg.), nor a thyreotropic fraction of the anterior-pituitary (16 mg.), nor injections of water (0·2 ml.), produced these histolytic changes in the cloacal region of ammocoetes.

It is of particular interest to note that those cells which have been shown to respond to the injection of hormone preparations, occupy similar positions in the cloacal regions of ammocoetes and adult lampreys (Text-figs. 1, 2). Since metamorphosis involves structural modifications of the cloacal region, whereby the mesonephric ducts are separated from the rectum, one may speculate whether those cells which respond in the ammocoete are the same cells which respond in the adult, or whether the response.of these cells is conditioned by the position which they occupy in the body, and not by a primary determination of the cell structure during ontogeny.

Pituitary preparations were the only agents which were found to stimulate the cloacae of both larval and adult lampreys. However, the response of the adult cloacal tissues to testosterone and oestrone makes it difficult to decide whether pituitary preparations have a direct effect on these tissues, or an indirect effect through a stimulation of the gonads.

Gonadectomy and the injection of pituitary preparations would decide this problem, but the size and position of the gonads makes gonadectomy impossible. Therefore one must rely on a histological examination of the gonads of injected animals to determine the role of the gonads in the cloacal response, although the extremely rapid response of the larval cloaca to pituitary preparations makes it improbable that the pituitary preparations act on the gonads of ammocoete larvae to produce this cloacal response.

A histological examination was made of injected animals and controls, in order to decide whether the hormone preparations which produced changes in the cloacal region, also affected the normal course of spermatogenesis.

An examination of the gonads of uninjected animals and animals injected with water showed a first appearance of sperms in the testes of a normal adult Lampetra fluviatilis on 14 March 1937. In a similar batch of animals kept by Miss U. Wykes in the Department of Zoology, Oxford, in 1938, the first sperms were found in the testis of a normal animal on 4 March. Therefore, it appears, that under the conditions used in these experiments, the normal male adult L. fluviatilis will not complete gametogenesis until the beginning of March.

Matured testes, however, were found at an earlier date in animals which had been injected with pituitary preparations or gonadial hormones. Free sperms were found in the testis and coelom of one injected animal as early as 3 February. This animal, whose gametogenesis was more than one month in advance of the normal testis development, had also a swollen cloaca as a result of an injection of 1 mg. of testosterone propionate. Two other animals were found to have sperms in their testes on 15 February. One of these had received ten injections, each of 10 mg., of preparation A.P. 6B. Both these animals had swollen cloacae, and histolytic changes were found in the cells of their mesonephric ducts. The testis of one control lamprey also matured earlier than those of the normal population. This animal had been injected with 0 ·1 ml. of sesame oil. When it was killed on 16 February its testes were found to contain sperms although no external modifications of the cloaca had occurred as a response to the injection. Therefore the early maturation of the gonads in those animals injected with pituitary preparations or the testicular hormone may not have been a specific response of the testis to these preparations.

Many other animals whose cloacae were swollen after injections of pituitary preparation A.P. 6B, testosterone or oestrone had testes which differed from those of the normal population. These testes were distinguished by the greater number of darkly staining nuclei which they contained (cf. Young & Bellerby, 1935). If these nuclei may be considered as stages in the normal spermatogenesis, it appears that the injection of hormone preparations may have accelerated this process. However, no normal animals were found to have testes containing similar nuclei. Therefore it is more probable that these nuclei are signs of an artificial degeneration of the testis.

Nevertheless, these experiments show that hormone preparations, which affect the cloacal region of adult lampreys, also have some effect on the testis.

The ovaries of injected female lampreys were also examined, but no distinct differences from the control ovaries were found.

The gonads of normal and injected ammocoetes were histologically examined, and it was found that the internal and external modifications of the cloaca, which appear after the pituitary injections, were unaccompanied by any apparent changes in the gonads of the injected animals. This observation would seem to indicate that the cloacal labia of ammocoetes respond directly to pituitary preparations, and the very rapid response of the cloaca supports this view (see P-542).

Previous investigators have reported that pituitary injections produced modifications in the gonads of ammocoetes. Young & Bellerby (1935) have described pycnotic nuclei in the testes of ammocoetes which had been injected with pituitary preparations for some weeks. Calvet (1932) has claimed an enlargement of the gonads of ammocoetes which had been placed in a solution containing pregnancy urine. It is possible, therefore, that in the present experiment the ammocoetes were killed before any changes in the gonads had become sufficiently marked to be apparent in a histological examination. The pituitary preparations produced an extremely marked cloacal response in 24 hours; injections extended over a longer period might produce noticeable changes in the gonads.

Ten ammocoetes from the river Sarno at Naples, measuring 115 mm. each, were injected with a solution of pituitary preparation A.P. 15 B. The dosage was arranged in order that it might be just below the threshold value for the production of swollen cloacae in ammocoetes. This was found by trial to be a dose containing $12$ mg. of preparation A.P. 15 B administered every third day. The experiment was concluded after one month and the gonads then histologically examined. Six of the ten ammocoetes were found to be males, but their testes presented a perfectly normal appearance. The female gonads were also indistinguishable from the ovaries of uninjected animals. There is therefore no evidence of a stimulation of the follicles by the injections as claimed by Calvet (1932).

In the river-lampreys which were kept under observation in 1937 it was noticed that maturity was attained by three distinct steps :

1. A modification of the fins. The development of the fins began before any marked changes in the gonads or cloacal region. Changes in the fins began at the end of February and continued until spawning.

2. Maturation of the gonads. Probably the early stages of spermatogenesis take place before modifications of the fins, but it was not until the end of March that late stages in spermatogenesis appeared in the testes of normal animals, together with a marked increase of the interstitial tissue.

3. A modification of the cloacal region. Finally, just prior to spawning, the cloacal labia became swollen, and ducts developed from the coelom to the exterior, allowing the genital products to escape.

The development of the secondary fin characters was not markedly affected by the injection of gonadial hormones or pituitary preparations. However, in view of Damas’ experiments, one cannot exclude the possible influence of the pituitary in the appearance of fin modifications.

It is difficult to determine the influence of the pituitary on gametogenesis. Damas has claimed that the injection of mammalian-pituitary preparations will accelerate gametogenesis, but in my experiments testosterone and sesame oil also appeared to produce a slight acceleration of gametogenesis. Possibly once spermatogenesis has commenced, it may be stimulated by any one of a number of substances within a specific range. This suggestion is supported by the work of Castelnuova (1937), who found that both male and female gonadial hormones, and pituitary preparations accelerated spermatogenesis in the carp.

The appearance of cloacal swellings and the formation of “ducts”, through which the genital products pass to the exterior, was greatly accelerated in the immature adult by the injection of gonadial hormones and pituitary preparations. Comparable changes even appeared in the ammocoete larvae after pituitary injections, although these changes do not normally appear in the larvae. This provides strong evidence for the endocrinal control of these characters in the normal mature adult.

Since both gonadial hormones and pituitary preparations accelerated the appearance of these characters in the adults, it is not clear whether the gonads or pituitary are the most probable source of an endocrine which influences the appearance of these characters at maturity. However, the cloacae of ammocoete larvae responded very rapidly to a small dose of a mammalian-pituitary preparation, but gave a weak response to testosterone propionate. This sudden and marked response of the ammocoete to a pituitary preparation but not to a testicular hormone indicates that the pituitary is the more probable source of an endocrine related to the cloacal region, but if this is the case the response of the adult to gonadial hormones is puzzling. The response of the cloaca may vary during the life of the animal, or possibly the adult cloaca is less selective in its capacity to respond to injected preparations.

Alternatively, the gonadial hormones may not act directly on the cloacal region, but may stimulate the release of some other hormone from the animal’s own pituitary. If the pituitary of ammocoetes is incapable of producing a hormone affecting the cloacal region, this hypothesis might explain the lack of any response in ammocoetes to the injection of male or female gonadial hormones.

The interest of these experiments, however, lies not only in the proof they provide of an endocrinal control of the secondary sexual characters in lampreys but also in a comparison with the endocrinal control of the sexual apparatus of higher forms. They provide the earliest instance in the phylogenetic series of the association of the pituitary gland of vertebrates with responses of the cloacal region. It is interesting to note that the response of this region at this early stage already includes vasodilatation, swelling, and epithelial changes, which are in some respects similar to those found in mammals.