In Vitro Effect of the Gonad of Helix Aspersa (Mollusca) on Galactogen Synthesis in the Albumen Gland of Either Mated Or Virgin Snails J Bride, L Gomot

In vitro effect of the gonad of Helix aspersa (Mollusca) on galactogen synthesis in the albumen gland of either mated or virgin snails J Bride, L Gomot

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J Bride, L Gomot. In vitro effect of the gonad of Helix aspersa (Mollusca) on galactogen synthesis in the albumen gland of either mated or virgin snails. Reproduction Nutrition Development, EDP Sciences, 1995, 35 (5), pp.559-567. hal-00899774

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In vitro effect of the gonad of Helix aspersa (Mollusca) on galactogen synthesis in the albumen gland of either mated or virgin snails

J Bride, L Gomot

Laboratoire de zoologie et embryologie, UFR sciences et techniques, place du Maréchal-Leclerc, 25030 Besançon cedex, France

(Received 26 September 1994; accepted 7 July 1995)

Summary ― The gonad of Helix aspersa contains a factor which can stimulate in a dose-dependent manner galactogen synthesis in albumen gland explants cultured in vitro. The stimulatory activity appears to be greater when the gonad is predominantly male than when it is predominantly female. The albumen gland of virgin snails does not respond in vitro to the gonadal influence. The receptivity of the albumen gland to the galactogen synthesis stimulating effect of the gonad is increased after the first and second mating. It decreases at the third mating in correlation with the increase of the albumen gland maturation index.

Helix aspersa / albumen gland / galactogen / gonad / mating

Résumé ― Effet in vitro de la gonade de Helix aspersa sur la synthèse de galactogène de la glande à albumen d’escargots vierges ou accouplés. La gonade de Helix aspersa contient un fac- teur capable de stimuler, d’une façon dépendante de la dose, la synthèse de galactogène dans des explants de glande à albumen cultivés in vitro. L’activité stimulante apparaît plus importante dans la gonade en phase mâle dominante que dans la gonade en phase femelle dominante. La glande à albumen d’escargots vierges ne répond pas à l’influence gonadique in vitro. La réceptivité de la glande à albumen à l’effet stimulant de la gonade sur la synthèse de galactogène est augmentée après le pre- mier et le deuxième accouplement. Elle diminue au troisième accouplement corrélativement à l’aug- mentation de l’indice de maturation glandulaire.

Helix aspersa / glande à albumen lgalactogène / gonade laccouplement INTRODUCTION !4C-glucose would be easier than ultrastructural observations. Secondly, this study In pulmonate molluscs, the albumen gland, was undertaken to check whether the effect demonstrated ultrastruc- one of the female accessory sex organs, gonadal secretes perivitelline fluid around the fertil- turally in some organites of cultured albumen and Courtot, cor- ized eggs (May, 1934). This fluid’s primary glands (Gomot 1979) constituent is galactogen (Goudsmit and responds to the stimulation of the Ashwell, 1965) which provides the main galactogen synthesis and whether varia- tions of this effect are correlated to energy source for the developing embryo physio- (Horstmann, 1965; Goudsmit, 1976). Growth logical stages of the sexual cycle. Investi- and differentiation of the albumen gland in gations were made in both virgin and mated snails. stylommatophoran snails and slugs are under the endocrine control of both the gonad (Abeloos, 1943; Laviolette, 1954; MATERIALS AND METHODS Sokolove et al, 1986) and the dorsal bodies (Wijdenes and Runham, 1976). With regard to the synthetic activity of the albu- Animals men gland, in vitro experiments demonstrated a direct endocrine control by factors produced either in the central nervous sys- One-month-old sexually immature snails were tem (Goudsmit, 1975, 1978) or in the non- raised individually in 500 ml plastic containers under constant nervous dorsal bodies (Van Minnen and temperature (20°C), photoperiod h 6 h and relative Sokolove, 1984). (18 L, D) humidity (95%). They were fed powdered food (UCAAB, Chierry, 02400 In Helix aspersa, the direct stimulation Chdteau-Thierry, France) ad libitum once a day. of organites implicated in the albumen gland These snails attained adult size and became sex- mature at 4 coincident with the secretion by the gonad was demonstrated ually months, upturning of the shell edge. Similarly, one-month- using an ultrastructural study of organ asso- old snails were raised in groups of 10 in 2 000 ml ciations cultured on a semi-solid medium plastic containers under the same conditions. (Gomot and Courtot, 1979). Castrations A jar filled with moist soil was provided and indicated that glass gonadal implantations for egg laying. Each snail was numbered with in addition to growth and differentiation, adhesive tape. The reproductive activity (copu- galactogen synthesis in the albumen gland lations, egg laying) was carefully monitored (once is stimulated by implantation of gonads in the morning, once in the evening). This allowed removed from active snails. In addition, it us to know exactly the reproductive stage of each animal. In this it was to choose was shown that implantation of gonads from way, possible pairs of snails to study the effect of either single hibernating snails caused an increase of or repeated mating on the in vitro response of albumen gland glycogen secretion whereas the albumen gland galactogen synthesis to the gonads from active snails caused a stimu- gonadal extracts. lation of de galactogen synthesis (Berset In H aspersa, egg laying is generally preceded Vaufleury et al, 1986). Therefore, it appears by multiple mating as it is reported in other species that the physiological state of implanted (Van Duivenboden and Ter Maat, 1985; Baur, gonads interferes with experimental results. 1988; Khan et al, 1990). Several data demonstrated the importance of as a stimulant of The aim of the in vitro in H mating present study the female sexual activity as compared to virgin aspersa was, first, to investigate a liquid snails (Van Duivenboden, 1983; Saleuddin etal, medium assay in which the determination of 1983, 1989; Khan et al, 1990; Saleuddin et al, the galactogen synthesis by incorporation of 1991). Tissue preparation Culture medium

The albumen gland was removed from virgin, sin- The liquid medium containing 14C-glucose as pre- gle-mated or repeatedly mated snails. After the cursor for galactogen synthesis was prepared as animal had been weighed, the albumen gland previously described (Bride et al, 1991 The cul- was removed and its maturation index (mi) was ture time was 24 h at room temperature in the calculated: dark.

albumen gland mi = wt of x 100 Determination of the galactogen wt of animal synthesis

Explants of albumen gland were then pre- The galactogen synthesis was determined by pared as previously described (Bride et al, 1991).). measuring the incorporation of !4C-glucose as Five samples were used for control conditions previously described (Bride et al, 1991).). and groups of 5 other samples for experimental conditions. The gonads were cleanly separated, teased Statistical analysis away from the underlying hepatopancreas with fine forceps and then rinsed in a saline solution (McCrone and Sokolove, 1979). Each gonad was The results were statistically tested in a one-way homogenized in a minimal volume (50 pl) of analysis of variance followed by the multiple range 0.1 M Hepes buffer, pH 7.4, then centrifuged at test of Newman and Keul (Zar, 1978) at a proba- 12 000 g for 10 min at 4°C. The supernatant con- bility level of 0.05. tained the gonadal extract of one animal equiv- alent (1 ae). For the study of the dose-response relation- RESULTS ship, gonads were removed from a population of adult reproductively active H aspersa in the dom- inant male after The phase egg-laying. super- Experiments in virgin snails natants were gathered together in a pool (R). Pooled gonads were also removed from 4-month- old snails virgin (V). Effect of gonadal extracts on the In paired snails, gonads and albumen glands albumen gland of virgin snails were removed within 6 h after copulation and for culture. The of immediately prepared stage Five 4.5-month-old snails were ran- differentiation of the was estimated virgin gonads by chosen. The influence of the careful examination under a microscope. In H domly pooled extract of either aspersa, the ovotestis first shows a male phase gonadal reproductively and contains mainly spermatozoa. This stage is active snails (R) or virgin snails (V) was gradually replaced by a phase of oogenesis investigated on the in vitro incorporation of including a primary vitellogenic stage character- 14C_glucose in galactogen synthesized by ized by small oocytes and a secondary vitel- explants of the albumen glands. Galacto- logenic stage containing large oocytes. gen was synthesized by individual albumen Each gonad of mated snails was individually glands at different basal rates as reported homogenized and the supernatant was divided in Goudsmit (1978) and Van Minnen et al in 2 of 0.5 ae. in each a 0.5 ae of parts Thus, pair, (1983). !4C-Glucose incorporation in the gonadal extract of one of the partners was added of R or V did not to autologous albumen gland explants and the presence vary significantly from the control basal other 0.5 ae to the explants from the second part- synthetic activity (table ner and vice versa (see fig 2 below). I). Thus, the albumen glands of 4.5-month- old virgin snails are not responsive to The albumen gland of single-mated gonadal influence in vitro. snail 1 was characterized by a mi of 3.05 whereas that of twice-mated snail 2 is (mi = 8.36). The Demonstration of the galactogen syn- larger galactogen synthesis in control medium was also different thesis stimulatory activity between the In twice-mated snail of the gonad of virgin snails partners. 2, the basal synthesis was 53% signifi- lower than control 1. The pooled extract of gonads of virgin snails cantly Nevertheless, the albumen of the mated (V) added to explants of albumen gland from gland repeatedly a reproductively active snail in the male phase after egg-laying (mi = 3.3) caused a significant increase of galactogen synthesis by 88% for 1 ae as compared to the control (fig ).1

Experiments in mated snails

Effect of gonads of reciprocally mated snails tested on their own albumen gland synthesis

Because a difference in the level of response of the albumen gland was found between the first-mated and the repeatedly mated snails, in our experiments we selected snails for which copulation occurred for the first time for snail 1 and for the second time without egg-laying for snail 2 (fig 2). snail 2 was more responsive to the 0.5 ae than the male gonad of the single-mated of gonadal extracts than that of single- snail 1. The gonad from snail 2 (0.5 ae) mated snail 1. Indeed, 0.5 ae of the male increased galactogen synthesis by only phase gonad of snail 1 caused an increase 110% compared control 1 and 183% com- of galactogen synthesis in albumen gland pared with control 2. from snail 2 of 610% compared with control In another experiment the pair consisted 2 and only of 280% in albumen gland from of snail 3 (which copulated for the first time snail 1 as compared with control 1. The in the second cycle of matings and egg-lay- gonad from twice mated snail 2 in the first ing, after emptying its albumen gland and vitellogenic female stage was less active gonad by egg-laying) and snail 4 which copulated for the third time in its first cycle with- the sexual cycle. out laid having eggs. The above in vitro results do not agree Albumen gland mi of the partners were with observations in basommatophorans different: 4.65 for the first-mated snail 3 in where castration experiments have indi- male phase, and 11.2 for the repeatedly cated that neither the development nor the mated snail 4 in second vitellogenic female activity of the accessory sex organs is under phase. The basal activity (control 4) of the endocrine control of the gonad (Brisson, large albumen gland from snail 4 was signif- 1971; De Jong-Brink et al, 1979; Vianey- icantly 3 times lower (p < 0.01) than that from Liaud, 1979). snail 3 (control 3). Only the minute gland from The results demonstrate that the level of the first-mated snail 3 was stimulated the by in vitro stimulation of albumen gland syn- 0.5 ae of its male phase gonad (3) which thetic activity by crude saline gonadal increased 68%. The galactogen synthesis by extracts depends on the receptivity of the albumen from snail 4, which was large gland albumen gland and on the differentiation mated 3 times, did not respond to the stimu- stage of the gonad. These 2 parameters of the male from snail latory activity gonad vary with the reproductive stages of the 3 (0.5 ae). The extract of the gonad from snails. Three stages of reproductive activity snail in second of the 4, vitellogenic stage have been considered in H aspersa, corre- female phase, did not significantly stimulate sponding to virgin, single-mated and repeat- the sensitive albumen gland from snail 3 or edly mated animals. the large albumen gland from snail 4.

Dose-response relationship of the Albumen gland receptivity effect of pooled gonadal extract and reproductive stage of the snail on in vitro galactogen synthesis The albumen gland from isolated virgin ani- The influence of aliquots of gonadal extract mal does not respond to the stimulatory R on the galactogen synthesis of the albu- activity of the gonadal extract. Two hypo- men gland (mi = 8.6) removed the day after thesis may be proposed concerning this the first mating was demonstrated (fig 3). observation: The minimum dose to elicit a stim- significant 1) The receptors implied in the stimulatory ulation over the control is 1 ae which activity of the gonadal extract are not acti- increased the !4C-glucose incorporation by vated, due to the lack of mating. 50% compared with the control. The dose to As it was demonstrated in Helisoma obtain a maximum response was 2 ae which 2) duryi and Saleuddin, we have caused a rise of galactogen synthesis by (Miksys 1987), observed that the albumen of iso- 102% compared with the control. glands lated virgin animals are more developed than in reproductive snails. A large amount of secretion stored in the albumen DISCUSSION AND CONCLUSION gland may explain the lack of galactogen synthesis stimulation we observed. This has been A point illustrated by this study is that the previously described in Lymnaea stagnalis gonad of adult active H aspersa appears to (Wijdenes et al, 1981 ), Limax maximus (Van contain a factor which exerts a direct stim- Minnen et al, 1983) and H duryi (Miksys and ulatory activity on galactogen synthesis of Saleuddin, 1985), where a large quantity of albumen glands in the appropriate stage of secretory material in albumen gland may inhibit the responses to galactogen synthe- more active in the presence of extract of sis stimulating factors from parts of the brain. male phase gonad in spermiogenesis (at the first than under the influence of After mating, there is a marked difference mating) in the level of the stimulation observed in vitro extract of vitellogenic female phase gonad between the albumen gland from single-mated (second and third mating). This difference of and repeatedly mated snails. At the second influence between predominantly male and female was also mating, the in vitro stimulation of the albumen predominantly gonads demonstrated in the in vitro of is than in study polysac- gland galactogen synthesis higher charide in the oviduct of snails mated either once or three times. The synthesis H aspersa (Bride and Gomot, 1988). The reason for first and the second the recep- mating promote these differences is not known. Neverthe- tivity of the albumen gland for the in vitro stim- less, they can be correlated with previous ulation of galactogen synthesis by the gonadal studies on implantation with male gonads extract. It can be extrapolated that the activa- in H aspersa de et al, tion of the (Berset Vaufleury galactogen synthesis machinery and in where would be caused the transfer of and 1986) slugs (Laviolette, 1954) by sperm implantation of gonads at the end of the male secretions from the Duiv- partner (Van spermiogenesis caused albumen gland and enboden, which have a direct stimula- 1983), common duct enlargement whereas there effect on the albumen and tory gland (Bride was no response to implantation of gonads 1991 At the third the Gomot, b). mating, large that had not begun spermiogenesis. In virgin albumen becomes in vitro gland unresponsive H aspersa, the gonads which elicit galacto- to the of the extract. stimulatory activity gonadal gen synthesis stimulating activity are full of Moreover, its low basal synthetic activity (con- spermatozoa and contain only previtel- trol that the in vivo level of value) suggests logenic oocytes. This was also observed in has also decreased. The galactogen synthesis H duryi, whose vitellogenesis was arrested absence of a significant effect of the gonadal in virgins because of the inactivity of the extract on the synthetic activity of the large dorsal bodies (Saleuddin et al, 1983). indicates that the of the secre- gland storage In conclusion, these results indicate the from of tory product arising synthesis galac- possibility of using this albumen gland bioas- togen after (Bride et al, 1991; Bride mating say to test the effect of fractions isolated by and account for the lack Gomot, 1991 a,b) may chemical extraction of the gonad. It also of stimulation for further of synthesis galacto- raises questions related to the cause of the gen. As observed in L stagnalis (Wijdenes et evolution of albumen gland receptivity with al, and L maximus Minnen et al, 1981) (Van respect to the role of repeated matings pre- the of the albu- 1983), quantitative response ceding egg-laying and the existence of a men gland of H aspersa can change consid- cycle in the production of gonadal endocrine erably with the weight of the gland, or mi, which factor(s) with respect to the correlation varies in different of the sexual stages cycle. between: a) spermiogenesis and the presence of a gonadal galactogen synthesis stimulatory substance; and b) vitellogene- Relation between the stage sis and the disappearance of this galactogen of differentiation of the gonad synthesis stimulatory substance. and the effect of gonadal extract on galactogen synthesis ACKNOWLEDGMENTS The nature of the stimulatory gonadal factor is unknown. 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