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Comparative study of the sterol composition of the digestive gland and the gonad of Sepia officinalis L. (mollusca, cephalopoda)
Comp. Biochem. PhysioL Vol. 83B, No. 3, pp. 599-602, 1986
0305-0491/86 $3.00 +0.00 ~3 1986 Pergamon Press Ltd
Printed in Great Britain
COMPARATIVE S T U D Y OF THE STEROL COMPOSITION OF THE DIGESTIVE G L A N D A N D THE G O N A D OF SEPIA O F F I C I N A L I S L.
(MOLLUSCA, CEPHALOPODA) BERNARD BLANCHIER,* EVE BOUCAUD-CAMOU*'~ a n d PIERRE SILBERZAHN~ *Laboratoire de Zoologie et ~Laboratoire de Biochimie, Universit6 de Caen, 14032 Caen Cedex, France (Tel: 31-94-81-40)
Abstraet--l. The sterol composition of the digestive gland and the gonad of Sepia oflicinalis L. was investigated by GC and GC-MS. 2. The same sterols were recognized in both organs, cholesterol being the major component of the sterol mixtures. However, quantitative differences appeared between the sterol composition of the digestive gland and the gonad. 3. The sterol mixtures of the digestive gland and the gonad of immature and mature females and males of various origins were compared. Quantitative changes in the sterol composition of the gonad were related to sexual maturity whereas the sterol composition of the digestive gland appeared linked to the diet.
INTRODUCTION
Cholesterol has long been reported to be the only sterol o f cephalopods. However, several recent studies using gas c h r o m a t o g r a p h y (GC) a n d mass spect r o m e t r y (MS) revealed t h a t the sterol c o m p o s i t i o n o f various c e p h a l o p o d s seems actually m o r e complex (Voogt, 1973; Idler et al., 1978; A p s i m o n a n d Burnell, 1980; Sica, 1980; Ballantine et al., 1980). All these results concern the whole animal, without reference to sex, except for the work o f Ballantine et al. (1980). Steroid h o r m o n e s (Nikitina et al., 1977) a n d steroid biosynthesis ( C a r r e a u a n d Drosdowsky, 1977) have been s h o w n to occur in the g o n a d o f cephalopods and, in a previous work o n Sepia officinalis (Blanchier a n d B o u c a u d - C a m o u , 1984), we noticed a n increase in the sterol level at sexual maturity. The digestive gland, which is the m a i n o r g a n o f digestion a n d storage ( B o u c a u d - C a m o u , 1973), has also been f o u n d to c o n t a i n sexual steroids (Nikitina et al., 1977). T h e present work was therefore designed to examine the sterol c o m p o s i t i o n o f the g o n a d as a function to sex a n d sexual m a t u r i t y a n d to c o m p a r e it with the digestive gland, b o t h in i m m a t u r e a n d m a t u r e females a n d males of Sepia officinalis L.
of maturity used were the occurrence of "smooth" oocytes in the ovarian sac for the females and of spermatophores in the Needham's sac for the males (Mangold-Wirz, 1963). Thirty-five immature females (70-430 g), 34 mature females (29001220 g), 17 immature males (105-275 g) and 75 mature males (1300980g) were analysed. The large number of mature males was related to the mode of capture of the animals (trapping with a female).
2. Methods The organs were analysed in batches from class-sized animals of the same origin, sex and state of gonadal maturation. The lipids were extracted and purified according to the method of Bligh and Dyer (1959). 0.1% propyl gallate was added to the extracting chloroform-methanol mixture to prevent oxidation. The lipid levels and the lipid composition of the digestive gland and the gonad have already been reported (Blanchier, 1981; Blanchier and Boucaud-Camou, 1984). The "free" (unesterified) sterol mixture was separated from the other lipids by preparative thin-layer chromatography (TLC) on silica gel. The "'total" sterol (unesterified and esterified) mixture was obtained after saponification of the lipid extract by 0.5 M KOH in 85% methanol for 6 hr at 60-70°C and separated by preparative TLC. ~4C-labelled cholesterol acetate was added to estimate the total yield. The trimethylsilylether derivatives of the sterols were subjected to gas chromatography (GC) and identified by comparing their retention time on different liquid phases with those of known standards. Cholestane was used as internal standard. GC was performed by a Pye Unicam chromatograph (model 104) fitted with a 1.5 m x 2 mm glass column packed with SE 30 at 1800230°C, 3-4°C/mn or OV 1. Additional evidence for the sterol identity was obtained by use of [Hewlett-Packard 5710 A 16 Micromass 305 aa (resolution 1000) gas chromatograph~mass spectrometry. The analyses were carried out at the "'Service Central d-'Analyses du C.N.R.S.] Universit6 Claude Bernard, Lyon". The mass spectra were analysed from the data of Beynon and William (1963), Knights (1967), Wyllie and Djerassi (1968).
MATERIALS AND METHODS
1. Materials One hundred and sixty-one immature and mature cuttlefishes (Sepia officinalis) of both sexes caught off the coasts of France (Mediterranean, Atlantic, Channel) by trawling or trapping were anaesthetized by ethanol, measured, weighed and promptly dissected. The digestive glands and the gonads were removed, weighed and deep frozen until analysis. Whenever immediate dissection could not be carried out, the whole animals were frozen. The two indices tTo whom correspondence should be addressed. 599
BERNARD BLANCHIERet al.
600
_/ Fig. 1. Chromatogram, obtained on SE 30, of the trimethylsilyl ether derivatives of the free sterols of the digestive gland of Sepia officinalis. 1-i0, peaks numbers related to the sterol components of Table 1. C, Cholestane (internal standard).
3. Nomenclature 22-Dehydrocholesterol: cholesta-5,22-dien-3fl-ol; cholesterol: cholest-5-en-3fl-ol; cholestanol: 50-cholest-5-en3#-ol; brassicasterol: 24-methylcholesta-5,22-dien-3fl-ol; desmosterol: cholesta-5,24-dien-3fl-ol; campesterol: 24methylcholest-5-en-3fl-ol; 24-methylene cholesterol: 24methylenecholest-5-dien-3fl-ol; stigmasterol: 24-ethyleholesta-5,22-dien-3fl-ol; sitosterol: 24-ethylcholest-5-en-3#-ol. RESULTS Figures 1 a n d 2 show the results of the c o l u m n c h r o m a t o g r a p h y of the free sterols of the digestive
5
2
Fig. 2. Chromatogram, obtained on SE 30, of the trimethylsilylether derivatives of the free sterols of the gonad of Sepia officinalis. 1-10, peaks numbers related to the sterol components of Table 1. C, Cholestane (internal standard). gland (Fig. l) a n d the g o n a d (Fig. 2). The n u m b e r of each peak is related to the sterol c o m p o n e n t s registered in T a b l e 1. Cholesterol, desmosterol, campesterol, stigmasterol (or its 24 isomer poriferosterol) a n d sitosterol were first recognized by their retention times compared to those of k n o w n standards. F u r t h e r i n f o r m a t i o n was o b t a i n e d from G C - M S o f the sterol mixtures. A mass spectrum c o r r e s p o n d i n g to peak No. 2 (on SE 30) showed a molecular ion at m/e 368 a n d the following m a i n f r a g m e n t ions (beyond 200): m/e 275,
Table 1. Sterol composition of Sepia offi'cinalis Relative % of total sterols % Sterols in RRT relative whole Sepia Peak No. Sterol components to cholestane Digestivegland Gonad (Voogt, 1973) 1 Monounsaturated C-26 1.05 0.2 0.7 2 24-Nor-cholest-5, 22-dien-3#-ol 1.07 3.2 0.7 0.5 3 Unknown 1.27 0.6 0.3 4 Unidentified C-27 (occelasterol?) 1.40 0.2 0-1 0 5 22-Dehydrocholesterol 1.48 2.5 0.6 0.5 6 Cholesterol 1.53 83.4 93.2 95.3 6 Cholestanol 1.53 trace trace 7 Desmosterol 1.58 3.4 3.6 1.5 8 Campesterot 1.66 (4.9 <0.5 0.6 8 24-Methylenecholesterol 1.66 1.1 9 Stigmasterol*+ 1.73 1.7 0.2 0.3 10 #-Sitosterolt 1.83 2 0.1 0 *Recognized only by GC. fOr poriferosterol.
Sterol composition of Sepia 257, 255, 247 and 213. This suggests a diunsaturated C-26 sterol such as 24-norcholest-5,22 dien-3fl-ol (Idler et al., 1970). A mass spectrum corresponding to peak No. 5 gave a molecular ion (M +) at m/e 384 and fragment ions at m/e 369, 366, 351,300, 271,255, 217 and was identified at that of 22-cholesta-5,22-dien-3fl-ol. A mass spectrum corresponding to peak No. 6 gave a molecular ion (M ÷) at m/e 368 and also displayed fragment ions at m/e 301,275, 255, 231 and was identified as that of cholesterol. In this spectrum, we found major fragments at m/e 388. 255. 233 and 215 from which we concluded that a small amount of cholestanol accompanies cholesterol. A mass spectrum corresponding to peak No. 7 gave a molecular ion (M ÷) at m/e 304 and the following fragment ions at m/e 369, 366, 252, 300, 299, 273, 271 and 229 and was identified as desmosterol. A mass spectrum corresponding to peak No. 8 showed two molecular ions (M ÷) at m/e 400, characteristic of a C-28 monounsaturated sterol, and (M ÷) at rn/e 398, indicating a C-28 diunsaturated sterol, and fragment ions at m/e 385, 315, 314, 289, 273,271, 255, 231,215 and 213. This mass spectrum was found to correspond to 24-methylenecholesterol and to campesterol. A molecular ion (M ÷) at rn/e 372, obtained from peak No. 1, could not be identified in the literature. It could eventually correspond to a monounsaturated C-26. In Table 1, we show the different sterols identified in the digestive gland and the gonad of Sepia officinalis. The average percentage of the sterol components were calculated on the basis of the chromatograms and compared to the composition given by Voogt (1973) for the whole animal. We can see that the digestive gland and the gonad displayed the same sterol composition: nine components have been recognized; cholesterol, which is the major constituant, 24-nor-cholest-5,22-dien-3fl-ol, 22-dehydrocholesterol, cholestanol, desmosterol, campesterol, 24-methylenecholesterol, stigmasterol (or its 24 epimer, poriferosterol), sitosterol. A further two have been partially identified: a monounsaturated C-26, and a C-27 which, from its retention time and data in the literature could be occelasterol (Kobayashi and Mitsuhashi, 1974). While the qualitative distribution of sterols is the same for digestive gland and gonad, the two organs differ in the relative amounts present. Cholesterol was always the main component, yielding less than 84% in the digestive gland but 93% in the gonad. Trailing far beyond was desmosterol, the second sterol component (around 4%), all the other sterols yielding less than 1% of the total sterol mixture. In the digestive gland, peak No. 8, corresponding to campesterol and 24-methylenecholesterol, yielded nearly 5% of the sterol mixture. The other sterols recognized yielded together 2 - 3 0 of the sterol mixture (Table 1). We also compared the composition of the "free" sterol mixtures to the "total" sterol mixtures in both organs, and did not notice any qualitative or quantitative differences.
601
Table 2. Relative percentage of free sterols in the digestive gland of Sepia officinalis 5
Peak numbers on SE 30 6 7 8 9 10
Females Immature Mature
1.7 2
85 84
2.9 3.5
4.8 4.5
1.2 2.3
2.5 1.6
Males Immature Mature
3.2 3.1
85.5 79
2.7 4.6
4.4 5.8
1.4 1.9
2 1.9
Table 3. Relative percentage of free sterols in the gonad of Sepia o~cinalis 5
Peak numbers on SE 30 6 7 8 9 10
Females Immature Mature
0.4 1.2
95.8 89.3
2.2 6.1
0.3 0.9
0.1 0.2
0.1 0.1
Males Immature Mature
0.2 0.8
95.5 92.5
1.6 4.5
0.2 0.6
0.3 0.4
0.1 0.2
Tables 2 and 3 show the relative percentage of the main sterols in the digestive gland (Table 2) and the gonad (Table 3) of immature and mature females and males. No marked sexual differences in the sterol composition occur either in the digestive gland or in the gonad. However, we noticed that there was a little more cholesterol in the mature testis than in the mature ovary and, conversely, more desmosterol in the ovary than in the testis. These differences are linked to the marked changes that occur at sexual maturity: we observed an increase of desmosterol and a concomitant decrease in cholesterol. These changes are more marked in the female than in the male, and explain the slight difference occurring in mature gonads. In the digestive gland, there were no obvious differences related to sexual maturity but we did notice large fluctuations from one batch of individuals to another. In the gonad, sterol composition remained constant between batches. DISCUSSION AND CONCLUSION
As far as the main components are concerned, the sterol compositions of Sepia officinalis digestive gland and gonad appear very close to that found by Voogt (1973) in the whole animal. However, some minor components had not been detected by this author. We recognized 24-nor-cholest-dien-3fl-ol, cholestanol and sitosterol in the sterol mixture. Our results agree with the few data available on other Coleoidea: Eledone aldrovandi and Octopus vulgaris (Voogt, 1973), Loligo vulgaris (Sica, 1980), lllex illecebrosus (Apsimon and Burnell, 1980) and Spirula spirula (Ballantine et al., 1981). All these authors found cholesterol to be the major component (85-95% of the sterol mixture). Cholestanol has been detected in I. illecebrosus. L. vulgaris and S. spirula. The sterols recognized in L illecebrosus by Apsimon and Burnell (1980) are the same as those we found in S. officinalis. Isofucosterol and brassicasterol (which we did not find) were detected in S. spirula by Ballantine et al. (1981). Actually, the sterol composition turns out to be not so "simple" as previously
BERNARD BLANCHIER el al.
602
thought, but, in contrast with cholesterol, the minor components occur in such small quantities that they are difficult to detect without special techniques and we imagine that further work will extend the list of sterols recognized in cephalopods. By comparing the sterol composition of the digestive gland and the gonad to the results obtained by Voogt from the whole animal (Table 1), we can see that the sterol composition of the gonad is very similar to that of the whole body. On the other hand, the digestive gland contains less cholesterol and more minor sterols. Moreover, this organ displays rather large fluctuations in the various sterol percentages. In Sepia officinalis, the digestive gland is the main organ of digestion and storage (Boucaud-Camou, 1973), so, it is likely that the changes in sterol composition are related to the diet. This supports the hypothesis of an exogenous origin for many of the sterols found in cephalopods (Zandee, 1967; Voogt, 1973, 1983). While, as we saw, the gonads of Sepia officinalis have a sterol composition close to that of the whole body, we noticed some marked changes related to sexual maturity: the cholesterol level decreases, while the levels of desmosterol and 22-dehydrocholesterol increase. The percentage is low, but, if we take into account the enlargement of the gonad (especially the ovary) and the sterol increase in the lipid content of the maturing animal (Blanchier and BoucaudCamou, 1984), the cholesterol content is seen to be multiplied by 30 and the desmosterol content by 100. One may wonder if the food can supply this considerable increase. Possibly, cholesterol and desmosterol could be partly synthesized from other sterols (stigmasterol, sitosterol and campesterol) or from mevalonate, although this last synthesis is very low (Voogt, 1973; Carreau, 1974). Much of the cholesterol must be involved in the large amounts of membrane produced during gametogenesis (Blanchier and Boucaud-Camou, 1984) but it is not excluded that some may be linked to steroid synthesis, thus explaining the decrease in cholesterol at gonadal maturity. Steroid biosynthesis from cholesterol has been demonstrated in the gonads of Sepia officinalis by Carreau and Drosdowsky (1977), suggesting that steroid hormones are involved in gonadal development in cephalopods. Acknowledgements--We would like to thank Dr R. Boucher-Rodoni and Dr S. von Boletzky for providing Sepia, Dr K. Mangold for reading the manuscript, Dr Zwingelstein for valuable suggestions and welcome in his laboratory and Mrs A. M. Renou for help with the figures. This work was supported by CNRS (U.A. 679 and U.A. 609) and DGRST (a research studentship for Dr Blanchier).
REFERENCES
Apsimon J. W. and Burnell D. J. (1980) Sterols from the squid lllex illecebrosus (Mollusca, Cephalopoda). Comp. Biochem. Physiol. 65B, 567-570.
Ballantine J. A., Robert J. C. and Morris R. J. (1980) The sterols of Crustaceans: decapods (sub-order Macrura). Comp. Biochem. Physiol 67B, 75-79. Ballantine J. A., Robert J. C. and Morris R. J. (1981) Sterols of the Cephalopod Spirula spirula. J. mar. Biol. Ass. 61, 843 850. Beynon J. H. and Williams A. (1963) Mass and Abundance Tables for the Use in Mass Spectrometry. Elsevier, New York. Blanchier B. (1981) Etude des lipides totaux et des st~roides dans la glande digestive et la gonade chez la Seiche Sepia officinalis L. (Mollusque C6phalopode). Thdse Doct. Specialit6, Universit~ de Caen. Blanchier B. and Boucaud-Camou E. (1984) Lipids in the digestive gland and the gonad of immature and mature Sepia officinalis (Mollusca: Cephalopoda). Mar. Biol. 80, 39-43. Bligh E. G. and Dyer W. J. (1959) A rapid method of the total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911-917. Boucaud-Camou E. (1973) Etude de l'appareil digestif de Sepia officinalis L. (Mollusque C6phalopode). Essai d'analyse exp6rimentale des ph6nomdnes digestifs. Thdse Doct. Sci. Nat., Universit6 de Caen. Carreau S. (1974) Mise en 6vidence biochimique des enzymes st6roidogdnes chez le mollusque C6phalopode Sepia officinalis L. Thdse Doct. Specialit& Universit6 de Caen. Carreau S. and Drosdowsky M. (1977) The in vitro biosynthesis of steroids by the gonad of the cuttlefish (Sepia officinalis L.) Gen. comp. Endocrin. 33, 554-565. Idler D. R., Wiseman P. and Safe L. M. (1970) A new marine sterol, 22-trans-24-norcholesta-5,22-dien-313-ol. Steroids 16, 451-461. Idler D. R., Khalil M. W., Brooks C. J. W., Edmonds C. and Gilbert J. D. (1978) Studies of sterols from marine mollusks by gas chromatography and mass spectrometry. Comp. Biochem. Physiol 59B, 163-167. Knights B. A. (1967) Identification of plant sterols using combined GLC: mass spectrometry. J. Gas Chromatog. 5, 273-282. Kobayashi M. and Mitsuhashi H. (1974) Marine sterols. V. Isolation and structure of occelasterol, a new 27-norergostane-type sterol, from an Annelida, Pseudopotamilla occelata. Steroids 24, 399-410. Mangold-Wirz K. (1963) Biologie des Cbphalopodes benthiques et nectoniques de l a m e r catalane. Vie et Milieu 13 (suppl.). Nikitina S. M., Savchenko O. N., Kogan M. E. and Ezhkova N. S. (1977) Extraction of progesterone, testosterone and oestrogens from the tissues of marine invertebrates. Zh. Evol. Biochim. Fiziol. XII, 443-447 (in Russian, English summary). Sica D. (1980) Sterols from some Molluscs. Comp. Biochem. Physiol. 65B, 407-410. Voogt P. A. (1973) Investigations of the capacity of synthesizing 3fl-sterols in Mollusca. X. Biosynthesis and composition of 3fl-sterols in Cephalopoda. Archs Int. Physiol. Biochim. 81, 401-407. Voogt P. A. (1983) Lipids: their distribution and metabolism. In The Mollusca (Edited by Wilbur K. M. and Hochachka P. W.), Vol. I, pp. 329 370. Academic Press, New York. Wyllie S. G. and Djerassi C. (1968) Mass spectrometry in structural and stereochemical problems. XXLVI. Mass spectrometric fragmentation typical of sterols with unsaturated side chain. J. org. Chem. 33, 305-313. Zandee D. I. (1967) Absence of cholesterol synthesis in Sepia officinalis L. Archs int. Physiol. Biochim. 75, 487~,91.
0305-0491/86 $3.00 +0.00 ~3 1986 Pergamon Press Ltd
Printed in Great Britain
COMPARATIVE S T U D Y OF THE STEROL COMPOSITION OF THE DIGESTIVE G L A N D A N D THE G O N A D OF SEPIA O F F I C I N A L I S L.
(MOLLUSCA, CEPHALOPODA) BERNARD BLANCHIER,* EVE BOUCAUD-CAMOU*'~ a n d PIERRE SILBERZAHN~ *Laboratoire de Zoologie et ~Laboratoire de Biochimie, Universit6 de Caen, 14032 Caen Cedex, France (Tel: 31-94-81-40)
Abstraet--l. The sterol composition of the digestive gland and the gonad of Sepia oflicinalis L. was investigated by GC and GC-MS. 2. The same sterols were recognized in both organs, cholesterol being the major component of the sterol mixtures. However, quantitative differences appeared between the sterol composition of the digestive gland and the gonad. 3. The sterol mixtures of the digestive gland and the gonad of immature and mature females and males of various origins were compared. Quantitative changes in the sterol composition of the gonad were related to sexual maturity whereas the sterol composition of the digestive gland appeared linked to the diet.
INTRODUCTION
Cholesterol has long been reported to be the only sterol o f cephalopods. However, several recent studies using gas c h r o m a t o g r a p h y (GC) a n d mass spect r o m e t r y (MS) revealed t h a t the sterol c o m p o s i t i o n o f various c e p h a l o p o d s seems actually m o r e complex (Voogt, 1973; Idler et al., 1978; A p s i m o n a n d Burnell, 1980; Sica, 1980; Ballantine et al., 1980). All these results concern the whole animal, without reference to sex, except for the work o f Ballantine et al. (1980). Steroid h o r m o n e s (Nikitina et al., 1977) a n d steroid biosynthesis ( C a r r e a u a n d Drosdowsky, 1977) have been s h o w n to occur in the g o n a d o f cephalopods and, in a previous work o n Sepia officinalis (Blanchier a n d B o u c a u d - C a m o u , 1984), we noticed a n increase in the sterol level at sexual maturity. The digestive gland, which is the m a i n o r g a n o f digestion a n d storage ( B o u c a u d - C a m o u , 1973), has also been f o u n d to c o n t a i n sexual steroids (Nikitina et al., 1977). T h e present work was therefore designed to examine the sterol c o m p o s i t i o n o f the g o n a d as a function to sex a n d sexual m a t u r i t y a n d to c o m p a r e it with the digestive gland, b o t h in i m m a t u r e a n d m a t u r e females a n d males of Sepia officinalis L.
of maturity used were the occurrence of "smooth" oocytes in the ovarian sac for the females and of spermatophores in the Needham's sac for the males (Mangold-Wirz, 1963). Thirty-five immature females (70-430 g), 34 mature females (29001220 g), 17 immature males (105-275 g) and 75 mature males (1300980g) were analysed. The large number of mature males was related to the mode of capture of the animals (trapping with a female).
2. Methods The organs were analysed in batches from class-sized animals of the same origin, sex and state of gonadal maturation. The lipids were extracted and purified according to the method of Bligh and Dyer (1959). 0.1% propyl gallate was added to the extracting chloroform-methanol mixture to prevent oxidation. The lipid levels and the lipid composition of the digestive gland and the gonad have already been reported (Blanchier, 1981; Blanchier and Boucaud-Camou, 1984). The "free" (unesterified) sterol mixture was separated from the other lipids by preparative thin-layer chromatography (TLC) on silica gel. The "'total" sterol (unesterified and esterified) mixture was obtained after saponification of the lipid extract by 0.5 M KOH in 85% methanol for 6 hr at 60-70°C and separated by preparative TLC. ~4C-labelled cholesterol acetate was added to estimate the total yield. The trimethylsilylether derivatives of the sterols were subjected to gas chromatography (GC) and identified by comparing their retention time on different liquid phases with those of known standards. Cholestane was used as internal standard. GC was performed by a Pye Unicam chromatograph (model 104) fitted with a 1.5 m x 2 mm glass column packed with SE 30 at 1800230°C, 3-4°C/mn or OV 1. Additional evidence for the sterol identity was obtained by use of [Hewlett-Packard 5710 A 16 Micromass 305 aa (resolution 1000) gas chromatograph~mass spectrometry. The analyses were carried out at the "'Service Central d-'Analyses du C.N.R.S.] Universit6 Claude Bernard, Lyon". The mass spectra were analysed from the data of Beynon and William (1963), Knights (1967), Wyllie and Djerassi (1968).
MATERIALS AND METHODS
1. Materials One hundred and sixty-one immature and mature cuttlefishes (Sepia officinalis) of both sexes caught off the coasts of France (Mediterranean, Atlantic, Channel) by trawling or trapping were anaesthetized by ethanol, measured, weighed and promptly dissected. The digestive glands and the gonads were removed, weighed and deep frozen until analysis. Whenever immediate dissection could not be carried out, the whole animals were frozen. The two indices tTo whom correspondence should be addressed. 599
BERNARD BLANCHIERet al.
600
_/ Fig. 1. Chromatogram, obtained on SE 30, of the trimethylsilyl ether derivatives of the free sterols of the digestive gland of Sepia officinalis. 1-i0, peaks numbers related to the sterol components of Table 1. C, Cholestane (internal standard).
3. Nomenclature 22-Dehydrocholesterol: cholesta-5,22-dien-3fl-ol; cholesterol: cholest-5-en-3fl-ol; cholestanol: 50-cholest-5-en3#-ol; brassicasterol: 24-methylcholesta-5,22-dien-3fl-ol; desmosterol: cholesta-5,24-dien-3fl-ol; campesterol: 24methylcholest-5-en-3fl-ol; 24-methylene cholesterol: 24methylenecholest-5-dien-3fl-ol; stigmasterol: 24-ethyleholesta-5,22-dien-3fl-ol; sitosterol: 24-ethylcholest-5-en-3#-ol. RESULTS Figures 1 a n d 2 show the results of the c o l u m n c h r o m a t o g r a p h y of the free sterols of the digestive
5
2
Fig. 2. Chromatogram, obtained on SE 30, of the trimethylsilylether derivatives of the free sterols of the gonad of Sepia officinalis. 1-10, peaks numbers related to the sterol components of Table 1. C, Cholestane (internal standard). gland (Fig. l) a n d the g o n a d (Fig. 2). The n u m b e r of each peak is related to the sterol c o m p o n e n t s registered in T a b l e 1. Cholesterol, desmosterol, campesterol, stigmasterol (or its 24 isomer poriferosterol) a n d sitosterol were first recognized by their retention times compared to those of k n o w n standards. F u r t h e r i n f o r m a t i o n was o b t a i n e d from G C - M S o f the sterol mixtures. A mass spectrum c o r r e s p o n d i n g to peak No. 2 (on SE 30) showed a molecular ion at m/e 368 a n d the following m a i n f r a g m e n t ions (beyond 200): m/e 275,
Table 1. Sterol composition of Sepia offi'cinalis Relative % of total sterols % Sterols in RRT relative whole Sepia Peak No. Sterol components to cholestane Digestivegland Gonad (Voogt, 1973) 1 Monounsaturated C-26 1.05 0.2 0.7 2 24-Nor-cholest-5, 22-dien-3#-ol 1.07 3.2 0.7 0.5 3 Unknown 1.27 0.6 0.3 4 Unidentified C-27 (occelasterol?) 1.40 0.2 0-1 0 5 22-Dehydrocholesterol 1.48 2.5 0.6 0.5 6 Cholesterol 1.53 83.4 93.2 95.3 6 Cholestanol 1.53 trace trace 7 Desmosterol 1.58 3.4 3.6 1.5 8 Campesterot 1.66 (4.9 <0.5 0.6 8 24-Methylenecholesterol 1.66 1.1 9 Stigmasterol*+ 1.73 1.7 0.2 0.3 10 #-Sitosterolt 1.83 2 0.1 0 *Recognized only by GC. fOr poriferosterol.
Sterol composition of Sepia 257, 255, 247 and 213. This suggests a diunsaturated C-26 sterol such as 24-norcholest-5,22 dien-3fl-ol (Idler et al., 1970). A mass spectrum corresponding to peak No. 5 gave a molecular ion (M +) at m/e 384 and fragment ions at m/e 369, 366, 351,300, 271,255, 217 and was identified at that of 22-cholesta-5,22-dien-3fl-ol. A mass spectrum corresponding to peak No. 6 gave a molecular ion (M ÷) at m/e 368 and also displayed fragment ions at m/e 301,275, 255, 231 and was identified as that of cholesterol. In this spectrum, we found major fragments at m/e 388. 255. 233 and 215 from which we concluded that a small amount of cholestanol accompanies cholesterol. A mass spectrum corresponding to peak No. 7 gave a molecular ion (M ÷) at m/e 304 and the following fragment ions at m/e 369, 366, 252, 300, 299, 273, 271 and 229 and was identified as desmosterol. A mass spectrum corresponding to peak No. 8 showed two molecular ions (M ÷) at m/e 400, characteristic of a C-28 monounsaturated sterol, and (M ÷) at rn/e 398, indicating a C-28 diunsaturated sterol, and fragment ions at m/e 385, 315, 314, 289, 273,271, 255, 231,215 and 213. This mass spectrum was found to correspond to 24-methylenecholesterol and to campesterol. A molecular ion (M ÷) at rn/e 372, obtained from peak No. 1, could not be identified in the literature. It could eventually correspond to a monounsaturated C-26. In Table 1, we show the different sterols identified in the digestive gland and the gonad of Sepia officinalis. The average percentage of the sterol components were calculated on the basis of the chromatograms and compared to the composition given by Voogt (1973) for the whole animal. We can see that the digestive gland and the gonad displayed the same sterol composition: nine components have been recognized; cholesterol, which is the major constituant, 24-nor-cholest-5,22-dien-3fl-ol, 22-dehydrocholesterol, cholestanol, desmosterol, campesterol, 24-methylenecholesterol, stigmasterol (or its 24 epimer, poriferosterol), sitosterol. A further two have been partially identified: a monounsaturated C-26, and a C-27 which, from its retention time and data in the literature could be occelasterol (Kobayashi and Mitsuhashi, 1974). While the qualitative distribution of sterols is the same for digestive gland and gonad, the two organs differ in the relative amounts present. Cholesterol was always the main component, yielding less than 84% in the digestive gland but 93% in the gonad. Trailing far beyond was desmosterol, the second sterol component (around 4%), all the other sterols yielding less than 1% of the total sterol mixture. In the digestive gland, peak No. 8, corresponding to campesterol and 24-methylenecholesterol, yielded nearly 5% of the sterol mixture. The other sterols recognized yielded together 2 - 3 0 of the sterol mixture (Table 1). We also compared the composition of the "free" sterol mixtures to the "total" sterol mixtures in both organs, and did not notice any qualitative or quantitative differences.
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Table 2. Relative percentage of free sterols in the digestive gland of Sepia officinalis 5
Peak numbers on SE 30 6 7 8 9 10
Females Immature Mature
1.7 2
85 84
2.9 3.5
4.8 4.5
1.2 2.3
2.5 1.6
Males Immature Mature
3.2 3.1
85.5 79
2.7 4.6
4.4 5.8
1.4 1.9
2 1.9
Table 3. Relative percentage of free sterols in the gonad of Sepia o~cinalis 5
Peak numbers on SE 30 6 7 8 9 10
Females Immature Mature
0.4 1.2
95.8 89.3
2.2 6.1
0.3 0.9
0.1 0.2
0.1 0.1
Males Immature Mature
0.2 0.8
95.5 92.5
1.6 4.5
0.2 0.6
0.3 0.4
0.1 0.2
Tables 2 and 3 show the relative percentage of the main sterols in the digestive gland (Table 2) and the gonad (Table 3) of immature and mature females and males. No marked sexual differences in the sterol composition occur either in the digestive gland or in the gonad. However, we noticed that there was a little more cholesterol in the mature testis than in the mature ovary and, conversely, more desmosterol in the ovary than in the testis. These differences are linked to the marked changes that occur at sexual maturity: we observed an increase of desmosterol and a concomitant decrease in cholesterol. These changes are more marked in the female than in the male, and explain the slight difference occurring in mature gonads. In the digestive gland, there were no obvious differences related to sexual maturity but we did notice large fluctuations from one batch of individuals to another. In the gonad, sterol composition remained constant between batches. DISCUSSION AND CONCLUSION
As far as the main components are concerned, the sterol compositions of Sepia officinalis digestive gland and gonad appear very close to that found by Voogt (1973) in the whole animal. However, some minor components had not been detected by this author. We recognized 24-nor-cholest-dien-3fl-ol, cholestanol and sitosterol in the sterol mixture. Our results agree with the few data available on other Coleoidea: Eledone aldrovandi and Octopus vulgaris (Voogt, 1973), Loligo vulgaris (Sica, 1980), lllex illecebrosus (Apsimon and Burnell, 1980) and Spirula spirula (Ballantine et al., 1981). All these authors found cholesterol to be the major component (85-95% of the sterol mixture). Cholestanol has been detected in I. illecebrosus. L. vulgaris and S. spirula. The sterols recognized in L illecebrosus by Apsimon and Burnell (1980) are the same as those we found in S. officinalis. Isofucosterol and brassicasterol (which we did not find) were detected in S. spirula by Ballantine et al. (1981). Actually, the sterol composition turns out to be not so "simple" as previously
BERNARD BLANCHIER el al.
602
thought, but, in contrast with cholesterol, the minor components occur in such small quantities that they are difficult to detect without special techniques and we imagine that further work will extend the list of sterols recognized in cephalopods. By comparing the sterol composition of the digestive gland and the gonad to the results obtained by Voogt from the whole animal (Table 1), we can see that the sterol composition of the gonad is very similar to that of the whole body. On the other hand, the digestive gland contains less cholesterol and more minor sterols. Moreover, this organ displays rather large fluctuations in the various sterol percentages. In Sepia officinalis, the digestive gland is the main organ of digestion and storage (Boucaud-Camou, 1973), so, it is likely that the changes in sterol composition are related to the diet. This supports the hypothesis of an exogenous origin for many of the sterols found in cephalopods (Zandee, 1967; Voogt, 1973, 1983). While, as we saw, the gonads of Sepia officinalis have a sterol composition close to that of the whole body, we noticed some marked changes related to sexual maturity: the cholesterol level decreases, while the levels of desmosterol and 22-dehydrocholesterol increase. The percentage is low, but, if we take into account the enlargement of the gonad (especially the ovary) and the sterol increase in the lipid content of the maturing animal (Blanchier and BoucaudCamou, 1984), the cholesterol content is seen to be multiplied by 30 and the desmosterol content by 100. One may wonder if the food can supply this considerable increase. Possibly, cholesterol and desmosterol could be partly synthesized from other sterols (stigmasterol, sitosterol and campesterol) or from mevalonate, although this last synthesis is very low (Voogt, 1973; Carreau, 1974). Much of the cholesterol must be involved in the large amounts of membrane produced during gametogenesis (Blanchier and Boucaud-Camou, 1984) but it is not excluded that some may be linked to steroid synthesis, thus explaining the decrease in cholesterol at gonadal maturity. Steroid biosynthesis from cholesterol has been demonstrated in the gonads of Sepia officinalis by Carreau and Drosdowsky (1977), suggesting that steroid hormones are involved in gonadal development in cephalopods. Acknowledgements--We would like to thank Dr R. Boucher-Rodoni and Dr S. von Boletzky for providing Sepia, Dr K. Mangold for reading the manuscript, Dr Zwingelstein for valuable suggestions and welcome in his laboratory and Mrs A. M. Renou for help with the figures. This work was supported by CNRS (U.A. 679 and U.A. 609) and DGRST (a research studentship for Dr Blanchier).
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