/. Embryol. exp. Morph. Vol. 60, pp. 57-69, 1980 57 Printed in Great Britain © Company of Biologists Limited 1980
Hormonal control of head-wart development in the snail, Euhadra peliomphala
By NAOKUNI TAKEDA1 From the Department of Biology, Toho University
SUMMARY The terrestrial snail, Euhadra peliomphala, has a peculiar organ between the optic tentacles named the head-wart, which releases a sex pheromone just before courtship. The development of the head-wart was closely correlated with the sexual maturity of the snail. Castration led to the atrophy of the head-wart. Subsequent injection of hermaphrodite gland homogenate into the body cavity of castrated individuals induced the development of the head-wart. The peripheral regions of the acini in the hermaphrodite gland showed a positive reaction for 3yff-hydroxy steroid dehydrogenase. Furthermore, preliminary radioimmunoassay also showed that the hermaphrodite gland of the snail contained testosterone and estradiol. When the immature head-wart was cultured for 10 days in a medium containing testosterone and estradiol respectively, development of the head-wart was found only in mediumcontaining testosterone. From these results, it is concluded that head-wart development in the snail, Euhadra peliomphala, is under direct control from the hermaphrodite gland, probably by means of testosterone.
INTRODUCTION An irregular polygonal pattern, named the head-wart, exists between the optic tentacles in some terrestrial snails (Taki, 1935). In a previous paper, we demonstrated that the head-wart released a sex pheromone just before courtship (Takeda & Tsuruoka, 1979). The development of the head-wart seems to be closely correlated with the sexual maturity of the snail, because it parallels progress of maturation of the reproductive system. Our knowledge of the endocrinology of pulmonate gastropods has progressed considerably during recent years (Joosse, 1975; Boer & Joosse, 1975). Since the discovery of estrogen and progesterone in the ovaries of Pecten hericius (Boticelli, Hisaw & Wotiz, 1961) many steroid hormones have been detected in the reproductive systems of molluscs (Lehoux & Sandor, 1970; Longcamp, Lubet & Drosdowsky, 1974; Nomura, 1978; Takeda & Kobayashi, 1980). The pulmonate gonad has also been shown to synthesize steroids related to those of the vertebrate gonadal system (Gottfried & Dorfman, 1970a, b). Although the endocrine role of these steroid hormones has not been proved fully in pulmonates, the presence of these substances seems to be related to the reproductive function. In the stylommato- 1 Author's address: Department of Biology, Facutly of Science, Toho University, Funabashi, 274, Japan. 58 N. TAKEDA phoran Mollusca, many experiments have suggested that the growth and differentiation of the accessory sex organs, such as the albumen gland and the common duct, are under the hormonal control of the hermaphrodite gland, e.g. in the slugs, Limaxflavus (Abeloos, 1943; Laviolette, 1954) and Agriolimax reticulatus (Bailey, 1973; Runham, Bailey & Laryea, 1973) and in the snails Helix aspersa (Gomot, 1973, 1976) and Taphius giabratus (Harry, 1965). The head-wart is an accessory sexual character and the development of the head- wart appears to be under hormonal control by the gonad as are the other accessory sex organs. In the present paper we attempt to clarify the hormonal control of head-wart development in the snail, Euhadra peliomphala.
MATERIALS AND METHODS The Euhadra peliomphala used in these experiments are commonly distributed terrestrial snails in Japan. Castration. As the hermaphrodite gland is located in the apex of the shell, the apical part of the shell was crushed for castration. The hermaphrodite gland resembles bunches of grapes which are partially embedded in the hepatopan- creas. The hermaphrodite gland was removed with forceps after cutting the hermaphrodite duct and separating it from the hepatopancreas. As the castra- tion is highly lethal, the operation resulted in partial castration. The opening was sealed with Spongel (Sankyo). Control snails were sham operated. Injections of hermaphrodite-gland homogenate and steroid hormones. An extract of the hermaphrodite gland was made using 30 adult snails. Pieces of hermaphrodite gland (5 mg) were homogenized in isotonic salt solution yielding a total of 10 ml. Injections of 0-1 ml were made into the body cavity by means of hypodermic needle. Control snails were injected with physiological salt solution only. 100/tg of the steroid hormones, testosterone and estradiol, were dis- solved in one drop of acetone and ethyl alcohol respectively. Each was diluted with 100 ml of double distilled water. Four injections were given on days 7, 9, 11, and 13 after castration. 3/1-hydroxy steroid dehydrogenase (3/1-HSD) activity. Fresh-frozen sections (15-20/£m) were made with a cryostat. They were picked up on cover glasses and dried at room temperature. The following incubation medium was used: Dehydroepiandrosterone (2-8 mg/ml acetone) 0-5 ml; nicotinamide (5 mg/ml distilled water) 1-0 ml; nicotinamide adenine dinucleotide (0-8 mg/ml of 0-1 M phosphate buffer at pH 8-0) 7-5 ml; nitro-blue tetrazolium chloride (1 mg/ml distilled water) 1-0 ml; phenazine methosulfate (0-1 mg/ml distilled water) 0-1 ml. Control sections were treated with the same medium without the substrate, dehydroepiandrosterone. Sections were incubated at 37 °C for 3 h, fixed in 10 % formalin and then mounted in Kaiser's glycerin jelly. Radioimmunoassay. Preliminary radioimmunoassays for estradiol and testo- sterone in the hermaphrodite glands were carried out by the methods of Control of head-wart development in the snail 59 Nakamura, Shodono & Tanabe (1974) and Hirano, Nakamura & Tanabe (1978). [2,4,6,7-H3]estradiol (specific activity, 105 Ci/m-mole) and [1,2,6,7-H3]- testosterone (specific activity, 100 Ci/m-mole) were used as antigens. Rabbit anti-estradiol-6-(O-carboxymethyl) oxine bovine serum albumen was used at 1:9000 dilution. Rabbit anti-testosterone-11-hemisuccinate bovine serum albumen was used at 1:20000. The diluted antisera were mixed with the ether- extracted hermaphrodite glands and [2,4,6,7-H3]estradiol or [1,2,6,7-H3]- testosterone and the mixture was incubated at 4°C overnight. Separation of the free from bound steroid was done by adding a suspension of 0-024 % charcoal in phosphate buffer. The radioactivity was measured with a liquid scintillation counter (Packard). Organ culture of the head-wart. The snails were starved for 3 days before use. They were washed several times with sterilized water after removing the shells. A piece of the immature head-wart epidermis was cut out and immersed in sterile Hedon-Fleig salt solution (Lockwood, 1961). This isolated epidermis was placed on Maximov slides in sterile Hedon-Fleig salt solution and exposed to ultraviolet irradiation (50 cm from the source) for 2 min for each side. Each explant was divided into two to four parts. Each part was transferred into a glass-ring chamber containing Hedon-Fleig salt solution. This solution was replaced with the culture medium. The culture medium used was developed for the snail, Helix aspersa by Burch & Cuadros (1965). For the experiments pieces of epidermis were cultured in media containing hermaphrodite-gland homo- genate, testosterone or estradiol. Cultures were kept at 25 °C for 10 days. The medium was changed every 2 days. Histology of the head-wart: The head warts, following both injections and culture, were fixed with Bouin's fluid, embedded in paraffin wax and sectioned at 8 //m. The sections were stained with hematoxylin and eosin.
RESULTS As the head-wart attains full size with the progress of sexual maturation, the development of the reproductive system and the head-wart was compared. In the infantile stage, the reproductive system consists of a simple elongated tube, the proximal end of which is connected to a rudimentary hermaphrodite gland, and the distal part connected to the small penial sheath. The other accessory sex organs are still rudimentary or entirely lacking. During growth of the shell the reproductive system remains small. With the development of the reversed periostome, the growth of the reproductive system was rapidly completed. The abrupt growth and differentiation of the reproductive system was related to the maturation of the gonad; the development of the head-wart appears to be closely correlated with this sexual maturation. Following castration, many snails died within 7 days but about 30 snails out of the 250 operated individuals survived for nearly 3 months. After the 60 N. TAKEDA
Fig. 1. Effects of castration on the epithelial cells of the head-wart in the adult snail, Euhadra peliomphala. (A) Epithelial cells of the head-wart in a control snail. Scale 25 /tm. (B) Epithelial cells of the head-wart in a castrated snail. Scale 25 /tm. operation, no sexual excitement was observed in such snails, movement became sluggish and the snails tend to withdraw into the shell. The appetite of the snail, decreased and the body weight, including shell, was gradually reduced. However, behavioural activity recovered about 6 days after the operation. The swelling of the head-wart was no longer observed. A complete atrophy of the genital orifice was seen in some snails about 10 days after the operation. The epithelial cells of the head-wart underwent regressive changes following castration (Fig. 1). A few days after castration, the height of the cells and the nuclear volume decreased and this inactive appearance persisted. However, with the injection of hermaphrodite-gland homogenate, the long axis elongated and the nuclear volume increased so that by about 20 days after castration the head-wart had recovered completely to appear normal (Figs. 2 and 3). These castration experiments suggest that the swelling of the head-wart is intimately related to the function of the hermaphrodite gland and that the development of the head-wart is controlled by substances secreted from the hermaphrodite gland. 3/?-hydroxy steroid dehydrogenase is one of the most important enzymes in the steroidogenic pathway. As shown in Fig. 4, 3/?-hydroxy steroid dehydroge- nase activity was clearly detected in the hermaphrodite gland, especially in the Control of head-wart development in the snail 61
70 -
60
50
40
A T I I I I I I I I I I I 1 1 I 1 1 I I I I 1 I 1 I I I I I I I I I 1 I 1 10 15 20 25 30 Days after castration Fig. 2. Effects of castration and injection on the development of the epithelial cells of the head-wart in the snail, Euhadrapeliomphala. •, Control snails; O, castrated snails; ©, injection of hermaphrodite gland homogenate; •, injection of testos- terone; A, injection of estradiol; $., injection: See text.
\ i i i i I i I i i I i i i I i i i i I i i i I I i i i I i I I I I I I
Fig. 3. Effects of castration and injection on the nuclear volume of the epithelial cells of the head-wart in the snail, Euhadra peliomphala. #, Control snails; O, castra- ted snails; ©3 injection of the hermaphrodite gland homogenate; •, injection of testosterone; A, injection of estradiol;
EMB 60 62 N. TAKEDA
Fig. 4. Occurrence of histochemical activity of 3/?-hydroxy steroid dehydrogenase in the hermaphrodite gland in the snail, Euhadra peliomphala. A, Frozen section of the hermaphrodite gland, demonstrating the reaction for 3/?-hydroxy steroid dehydrogenase in the peripheral region of the acini. Arrow. Scale 25 /tm. B, Cross- section of the hermaphrodite gland, showing sperm and eggs with germinal vesicles. Scale 25 /tm.
Table 1. Detection of steroid hormones (testosterone and estradiol) in the herma- phrodite gland of the snail, Euhadra peliomphala. Experiments were performed in January which was the diapause period in all snails and the post-reproductive stage in adult snails
Hermaphrodite Snails glands* Testosteronef Esttadiol (g) (mg) (pg/mg tissue) Infantile (1-8) 131 2-70 3-82 Juvenile (5-8) 262 219 1 97 Adult (8 0) 650 0-20 1-30 * The total amount of 13 individuals. t Mean of the amount in 13 pooled gonads. Control of head-wart development in the snail 63
Fig. 5. Effects of hermaphrodite-gland homogenate on the development of the epithelial cells of the head-wart in the juvenile snail, Euhadia peliomphala in vitro. (1) Control, scale 25 /tm, (2) Hermaphrodite gland homogenate, see text, scale 25/mi. Cultures were performed for 10 days at 25 °C. cells at the periphery of each acinus. The hermaphrodite gland was shown to have the ability to synthesize steroid hormones. Furthermore, testosterone and estradiol were demonstrated by radioimmuno- assay within the hermaphrodite gland (Table 1). The amount of steroid hormones was much greater in infantile snails than in juvenile and adult snails. More estradiol was present than testosterone. The adult snails in the post-reproductive condition contained low levels of these steroid hormones. 5-2 64 N. TAKEDA
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FIGURE 6 Effects of steroid hormones (estradiol and testosterone) on the development of the epithelial cells of the head-wart in the juvenile snail, Euhadra peliomphala in vitro. (1) Control, scale 25 /tm. (2) Estradiol (50/tg/dl medium), scale 25 /*m. (3) Testos- terone plus estradiol (each 50 /*g/dl medium), scale 25 /tm. (4) Testosterone (50 [i%/ dl medium), scale 25 fim. Cultures were performed for 10 days at 25 °C. Control of head-wart development in the snail 65
Table 2. Effects of hermaphrodite-gland homogenate and steroid hormones {testosterone and estradiol) on the development of the epithelial cells of the head- wart in the snail, Euhadra peliomphala. As the nuclei during cultivation were approximately ellipsoid, the volume of each nucleus was calculated by the formula, V = nLl2/6. See Fig. 3
Length (/tm) , A ^ Nuclear volume Treatments Long axis Short axis (/t3) Hermaphrodite gland 68-8±15-4 5-6±0-8 171-5±570 homogenate Estradiol + Testosterone 48-8±6-3 6-5±l-3 153-8±43-2 Control 431 ±8-8 6-7±l-9 151-5±60-2
Testosterone 69-3 ±13-3 5-8 + 1-4 172-4 ±460 Estradiol 31-8 + 15-3 7-4±3-2 117-0±4M Control 35-8 ±10-3 6-8 + 1-6 119 3±46-5 Concentration of steroid hormones; 50/ As the hermaphrodite gland was shown to be able to synthesize steroid hor- mones, the biological activity of these hormones was examined. When testo- sterone was injected into the body cavity of castrated snails, the head-wart developed gradually and recovered from castration within a few days (Figs. 2 and 3). However, no clear effect of estradiol injection was found. These sub- stances were also injected into intact snails. The development of the head-wart was also stimulated by injection of hermaphrodite gland homogenate and testosterone but no effect was found following estradiol injection. These effects were not as clear as in castrated snails. When the immature head-wart was cultured in a medium containing herma- phrodite-gland homogenate, it developed enormously (Fig. 5). The epithelial cells of the head-wart cultured with testosterone developed similarly (Fig. 6). However, no effect was found in the head-wart cultured with estradiol. Anta- gonistic effects of estradiol and testosterone were found in the head-wart cultured with testosterone plus estradiol. The development of the head-wart was reflected not only in the nuclear volume change but also in the elongation of the epithelial cells. Following 10 days cultivation, the development of the head-wart was not as advanced as during the breeding season of the snails. These results are summarized in Table 2. From the results, it is suggested that the development of the head-wart is controlled by testosterone secreted from the hermaphrodite gland of the snail. 66 N. TAKEDA DISCUSSION The head-wart is present in the Pleurodontidae, genera Satsuma and Chlorites, and in the Cepolidae, genera Helicostyla, Aegista, Bradybaena, Euhadra, Doiicheulota and Trishoprita (Taki, 1935). Several species of the African genus Gymnarion carry on their head a similar organ called the frontal organ (Binder, 1969). These organs are only complete in adult snails during the mating season. Furthermore, their existence implies a very particular mating behaviour (Binder, 1977; Takeda & Tsuruoka, 1979). The complete development of these organs has been suggested to be correlated with that of the reproductive system and thus an external indicator of their endocrinological state. In some terrestrial pulmonate Stylommatophora, the growth and differentia- tion of the accessory sexual organs have been known to be under the hormonal control of the hermaphrodite gland. Castration of the adult slugs, Avion ater and Limax maximus (Abeloos, 1943; Laviolette, 1954) led to the regression of the common duct and the albumen gland. Castration of shell-bearing snails in which hermaphrodite glands are embedded in the hepatopancreas has been known to be highly lethal. In Euhadra peliomphala, the survival time was also short and the operation resulted in only partial castration. However, following even this partial castration, the head-wart failed to develop. As in many gland cells (Gabe & Arby, 1961), the increase and decrease in the nuclear volume of the head-wart epithelial cells is also considered to represent the increasing and decreasing activity of the head-wart. As the injection of the hermaphrodite- gland homogenate into castrated snails induced the development of the head- wart, it is suggested that the hormones involved with the development of the head-wart originate from the hermaphrodite gland. In Gymnarion, accidental castration also led to the atrophy of the frontal organ (Binder, 1969). From castration experiments it has been suggested that maintenance of the structural characteristics of active secretion in the multifid gland in the snail, Helix aspersa is partially dependent on a hormonal factor secreted from the hermaphrodite gland (Gomot, 1973, 1976). The hormonal role of the hermaphrodite gland in regulating accessory sexual gland development has therefore been established. These changes are similar to those of the accessory reproductive glands of mammals such as the prostate and seminal vesicles. However, no effect of castration on the growth of the accessory sexual organs was found in the snail, Bulinus truncatus (Brisson, 1971; de Jong-Brink, Borg, Bergamin-Sassen & Boer, 1979) and Australobis glabratus (Vianey-Liaud, 1972). There is tentative evidence that brain hormones may influence the reproduc- tive organs via the gonad. Bailey (1973) proved with organ culture that the cerebral ganglia and gonad in association, but not when cultured separately, produce hormones that affect the male line in the gonad and secreting cells in the prostate gland. In the snail, Lymnaea stagnalis, the dorsal body is known to produce a hormone that stimulates vitellogenesis and growth of the female Control of head-wart development in the snail 67 accessory sexual organs (Geraerts & Joosse, 1975; Geraerts & Algera, 1976). In the slug, Agliolimax reticulatus, dorsal body hormone also controls the growth and differentiation of the oviducal and albumen glands (Wijdenes & Runham, 1976). In these animals, the effect of the dorsal body hormone on the female organ could be mediated by the hermaphrodite gland. The detection of a steroid-synthesizing enzyme such as 3/?-hydroxy steroid dehydrogenase in the hermaphrodite gland in Euhadra peliomphala has shown that the synthesizing system for steroid hormones is present. In the pulmonate Basommatophora, the hermaphrodite gland is not known to have an endocrine function (Boer & Joosse, 1975; Joosse, 1975). However, recent experiments on Lymnaea stagnalis have shown that its hermaphrodite gland also possesses 3/?-hydroxy steroid dehydrogenase (de Jong-Brink, Boer & Schot, 1978). This probably indicates that the gonad is capable of producing steroid hormones, although the endocrine role has not yet been proved. Experimental evidence shows that the pulmonate hermaphrodite gland pro- duces one or two sexual hormones. However, the cells involved in the synthesis of these hormones are as yet not identified. Gottfried & Dorfman (1968, 1970 a, b) have shown that steroid hormones, related to the vertebrate gonadal hor- mones, are synthesized by the hermaphrodite gland of Ariolimax californicus. In this species, steroid hormones are implicated in modifying the eye-tentacle effect upon the hermaphrodite gland. In the slugs, Deroceras reticulatum and Limaxflavus, estrogen stimulated egg-laying in spite of the low rate of develop- ment, whereas androgen enhanced the rate of development rather than the number of eggs laid (Takeda, 1979). Furthermore, in Umax flavus, sperma- togenesis is controlled by testosterone secreted from the hermaphrodite gland (Takeda &Tejima, 1979). In the present experiments, it was demonstrated that the head-wart in the snail, Euhadra peliomphala, is the target organ of the steroid hormone, testos- terone, secreted from the hermaphrodite gland. It is interesting to note the resemblance between the roles of steroid hormones in vertebrates and in invertebrates. The author wishes to express his thanks to Prof. J. Ishida, Toho University, for his en- couragement of the present work, to former Prof. Iw. Taki, Hiroshima University, for his many helpful suggestions and encouragement, to Prof. Y. Tanabe and Dr T. 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