Cell Tiss. Res. 183, 25-32 (1977)
Cell and Tissue Research 9 by Springer-Verlag 1977
Ultrastructural Localization of Juvenile Hormone Biosynthesis by Insect Corpora Allata* S.S. Tobe Department of Zoology, University of Toronto, Toronto, Ontario, Canada A.S.M. Saleuddin Department of Biology, York University, Downsview, Ontario, Canada
Summary. The conversion of exogenous 3H-farnesenic acid to 3H-methyl farnesoate and 3H-C16 juvenile hormone (JH) has been followed in the corpus allatum (CA) cells of the desert locust Schistocerca gregaria by means of electron microscopic autoradiography. Aerobic and anaerobic 'chase' incubations have been used to modify the quantities of these three compounds within the CA cells. Under all incubation conditions, radiolabel is found associated almost exclusively with the subcellular m e m b r a n e s y s t e m s - smooth endoplasmic reticulum (SER) and Golgi elements - and with the mitochondria. CA cells are p r o b a b l y similar to vertebrate steroid-synthesizing cells in that the secretory product is synthesized in the SER and mitochondria. Radiolabel was found to be present in all cells of the CA suggesting that all cells are capable of at least the final two stages of J H biosynthesis (the esterification and epoxidation of 3H-farnesenic aid). This indicates that J H biosynthesis m a y be regulated through changes in the biosynthetic capabilities of individual cells rather than through changes in the total number of cells engaged in biosynthesis. Radiolabel was not observed to be associated with any distinctive cellular product, a result which provides additional evidence for the suggestion that the release of J H from the CA is governed by laws of simple physical diffusion. Key words: Corpus allatum - Juvenile hormone biosynthesis - Localization. Introduction The corpus allatum (CA) is the site of synthesis and release of the juvenile hormone (JH) in all insect species studied to date. In the desert locust, Schistocerca gregaria, the CA have been shown to synthesize and release C16JH (methyl 10,11-epoxySend offprint requests to: Dr. S.S. Tobe, Department of Zoology, University of Toronto, 25 Harbord St., Toronto, Ontario, Canada M5S 1A1
* Supported by operating grants from the National Research Council of Canada to SST and ASMS. 3H-farnesenic acid was supplied by the late Dr. A.F. White of the Unit of Invertebrate Chemistry and Physiology, A.R.C., University of Sussex. We thank Dr. G.E. Pratt for helpful discussions
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3,7,11-trimethyl-2,6-trans, trans-dodecadienoate) when m a i n t a i n e d in vitro in a defined tissue culture m e d i u m for p e r i o d s o f u p to 5 h ( P r a t t a n d Tobe, 1974; T o b e a n d Pratt, 1974 a; P r a t t et al., 1975 a). T h u s the C A o f locusts have the c a p a b i l i t y to synthesize C16JH f r o m only simple p r e c u r s o r s present in the defined m e d i u m . In a d d i t i o n , locust C A can be s t i m u l a t e d to p r o d u c e larger quantities o f C a6JH by the a d d i t i o n o f the e x o g e n o u s p r e c u r s o r farnesenic acid to the i n c u b a t i o n m e d i u m ( P r a t t a n d Tobe, 1974; T o b e a n d Pratt, 1974a). It has been shown t h a t the e x o g e n o u s farnesenic acid is c o n v e r t e d to C16JH b y esterification a n d e p o x i d a t i o n a n d t h a t these final two stages in J H biosynthesis are n o t rate-limiting in C A o f S. gregaria ( P r a t t a n d Tobe, 1974; P r a t t et al., 1975 b). Because farnesenic acid is so readily i n c o r p o r a t e d into J H b y locust CA, we h y p o t h e s i z e d t h a t the r a d i o l a b e l l e d f o r m o f this c o m p o u n d c o u l d be used to ascertain the subcellular c o m p a r t m e n t s in which at least the final two stages o f J H biosynthesis occur. To this end, we have d e v e l o p e d a m e t h o d o l o g y which permits retention o f significant a m o u n t s o f r a d i o l a b e l within the C A after p r e p a r a t i o n for light a n d electron m i c r o s c o p i c a u t o r a d i o g r a p h y a n d following a variety o f pulse-chase p r o c e d u r e s ( T o b e a n d P r a t t , 1976; T o b e et al., 1976). In the p r e s e n t p a p e r we r e p o r t o n the s u b c e l l u l a r l o c a l i z a t i o n o f r a d i o l a b e l l e d p r o d u c t s as d e t e r m i n e d by electron m i c r o s c o p i c a u t o r a d i o g r a p h y . W e have r e p o r t e d previously on the light m i c r o s c o p i c localiz a t i o n o f r a d i o l a b e l l e d p r o d u c t s ( T o b e a n d Pratt, 1976). Materials and Methods
Schistocercagregariawere reared after the method of Tobe and Pratt (1975). Animals used in this study were females 2, 4 and 6 days after fledging (CA inactive) and 8 and 11 days after fledging (CA active). Corpora allata were dissected and incubated in the presence of 3H-farnesenic acid (final concentration 20 I~M)as described previously (Tobe and Pratt, 1976; Tobe et al., 1976). With radiochemical procedures, we have previously shown that under standard aerobic conditions, more than 50 % of the radiolabel within the CA is present as C 16JH after 30 min. When this standard incubation is followed by a twenty minute aerobic chase, more than 70 % of the radiolabel in the CA is present as C16JH, whereas if the standard incubation is followed by a twenty minute anaerobic chase (under nitrogen), approximately 50 % of the radiolabel is present as methyl farnesoate (Tobe and Pratt, 1976). We have employed these three treatments to obtain CA with varying amounts of radiolabelled farnesenic acid, methyl farnesoate and C16JH for evaluation by electron microscopic autoradiography. After the appropriate treatment, CA were fixed by a modification of the method of Hirsch and Fedorko (1968). The fixative consisted of a freshly prepared mixture of 2 parts 1% osmic acid in 0.1 M cacodylate buffer (pH 7.4) and 1 part 2.5 glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). The CA were embedded in Araldite 502 : Epon 812. For autoradiography, thin sections, placed on collodion-coated slides and allowed to dry, were stained with uranyl acetate and lead citrate, coated with a thin layer of carbon and dipped in Ilford L-4 emulsion after the method of Salpeter et al. (1969). Sections were incubated for 6-8 weeks, developed in Kodak D19 and examined in a Philips EM 201. Loss of label during each fixation and embedding step was monitored by counting aliquots of the fixative, ethanols and embedding media; this loss averaged 4050 % of the total label initially present.
Results and Discussion T h e C A o f S. gregaria are similar in m o r p h o l o g y to those o f o t h e r locust species ( G u e l i n a n d D a r j o , 1974). T h e y are c h a r a c t e r i z e d by an extensive s m o o t h e n d o p l a s m i c r e t i c u l u m (SER), n u m e r o u s m i t o c h o n d r i a a n d a p r o m i n e n t nucleus. In
Localizationof JuvenileHormoneBiosynthesis
27
the CA cells ofS. gregaria, the mitochondria are large (Odhiambo, 1966), and small amounts of rough endoplasmic reticulum are present. In CA known to be synthesizing and releasing Ci6JH at a high rate (days 8-11 after fledging), numerous lysosomes, multivesicular bodies and Golgi elements also occur. Extensive extracellular stroma and axons, many containing neurosecretory granules, occur within the glands (Scharrer, 1964; Melnikova and Panov, 1975). Extracellular channels continuous with the gland's sheath afford communication with the haemocoel. The same system of extracellular channels carrying neurosecretory fibres occurs in the CA of Leucophaea maderae (Scharrer, 1964). The presence of abundant SER and mitochondria in CA cells has led many authors to suggest, by comparison with vertebrate steroid-synthesizing cells, that the synthetic product is lipoidal (see Fawcett et al., 1969). In S. gregaria the synthetic product is the sesquiterpenoid Ci6JH (Pratt and Tobe, 1974; Tobe and Pratt, 1974a, 1975) and, on this basis, it might be predicted that a significant proportion of the radiolabel would be associated with the SER and mitochondria. This is indeed the case; grains are localized almost exclusively on SER and occasional mitochondria and Golgi complexes (Figs. 1, 2). No difference in the distribution of radiolabel was observed between glands fixed immediately after 3H-farnesenic acid incubation and glands chased either aerobically or anaerobically before fixation. There is a difference in the total amount of label within the CA after these different treatments and this is reflected in the total number of grains observed by light microscopic autoradiography (Tobe and Pratt, 1976). Thus, although the total amounts of label observed within the CA after the three treatments may differ, the subcellular distribution of this label is similar in all cases. The use of these different treatments theoretically permitted the localization of specific metabolites in specific subcellular compartments, but since no differences in the distribution of label are apparent after these different treatments, we must conclude that the three metabolites which are being visualized by autoradiography are confined mainly to the SER and, to a lesser extent, to the mitochondria. Specific metabolites cannot be associated with specific organelles. Hammock (1975) reported that the final stage in JH biosynthesis, the epoxidation of methyl farnesoate, is largely confined to the microsomal fraction of the CA in Blaberus giganteus. This finding agrees well with the observed localization of radiolabel in the SER reported in the present paper. Two different types of SER can be observed in the CA cells of S. gregaria: concentric lamellar (Fig. 2) and vesiculate (Fig. 3). Label can be associated with both types. Both types have also been reported in the CA ofLocusta migratoria, although the functional difference between them remains unclear (Joly et al., 1968; FainMaurel and Cassier, 1969). These authors suggested that vesiculate SER is found in highly active glands whereas lamellar SER is found in inactive or involuting glands. Joly et al. (1968) further suggested that the fragmentation of the lamellar SER is important for the release of JH. However, our autoradiographic results do not support this contention since the label is not associated with any visible cellular product. The question of the number of cells actively engaged in the synthesis of JH has interested insect endocrinologists for some time. In a low magnification autoradiograph showing a number of different CA cells (Fig. 4), it can be seen that
Fig. 1. Autoradiograph of CA from 11 day S. gregaria showing grains on Golgi apparatus (arrow) and mitochondrion (white arrow). No chase. L lysosome, x 20,900 Fig. 2. Autoradiograph of CA from 11 day S. gregaria showing grains in concentric lamellae of smooth endoplasmic reticulum (SER). After 30min incubation in 3H-farnesenic acid, CA were chased aerobically for 20 min before fixation. T trachea. • 20,900
Localization of Juvenile Hormone Biosynthesis
29
Fig 3. Autoradiograph of CA from 8 day S. gregaria showing grains on vesicular SER. No chase. L lysosome, x 20,900 Fig. 4. Autoradiograph of CA from 11 day S. gregaria showing random distribution of grains. No chase. Arrows indicate Golgi apparatus. L lysosome; M mitochondrion. • 15,200
30
s.s. Tobe and A.S.M. Saleuddin
all cells contain grains. Therefore, all cells of the CA must be capable of at least esterification and epoxidation of 3H-farnesenic acid and this supports our previous light microscopic observation that all cells of the CA appear to be equally competent to effect the final two stages in JH biosynthesis (Tobe and Pratt, 1976). This result is significant in that the overall synthetic capacity of the CA is not regulated through the proportion of cells engaged in JH biosynthesis but rather through changes in the biosynthetic capabilities of individual cells. Guelin and Darjo (1974) reported that the CA cells of L. migratoria may function asynchronously and that thus not all cells may be equally capable of JH biosynthesis at any given time. It is conceivable that the secretory cells of the CA of S. gregaria undergo asynchronous cycles of JH biosynthesis but in the light of our radiochemical findings, which demonstrate large and predictable cyclical changes in JH biosynthesis in concert with the gonadotropic cycles, such asynchrony does not seem likely. Nonetheless, the methods used in the present study may not be sufficiently discriminating to permit the observation of different amounts of radiolabel within individual cells. Although total numbers of grains in individual cells were not determined, our previous light microscopic autoradiography indicated that the radiolabel is uniformly distributed throughout the glands, also suggesting that all CA cells are capable of effecting the final two stages in JH biosynthesis. It should be stressed that the use of 3H-farnesenic acid as a radiolabelled probe for the localization of JH biosynthesis reveals only the final two stages in the biosynthetic pathway. At present, the subcellular location of the early stages in JH biosynthesis is unknown although the synthesis of farnesyl pyrophosphate may follow the pathway described for vertebrate steroid-producing cells, i.e., SER and cytoplasmic matrix (Fawcett et al., 1969). An autoradiographic study of the early stages of JH biosynthesis may reveal areas of potential cytoplasmic compartmentalization which may be important in the regulation of overall JH biosynthesis by controlling the access of substrates to the appropriate enzyme compartments. As has been noted above, the radiolabel within the CA is present as 3H-Ca6JH and, to a lesser extent, as 3H-methyl farnesoate after different treatments. It is striking that the grains do not appear to be associated with any distinctive secretory product. It has been demonstrated that significant amounts of 3H-C16JH are present in the incubation medium within 10 min of the addition of 3H-farnesenic acid (Tobe and Pratt, 1974 a) and this observation led in part to the suggestion that the release of JH from the CA is governed by laws of simple physical diffusion; JH is released soon after it is synthesized (Tobe and Pratt, 1974 b). In view of the rapidity of release of the hormone, it is not surprising that the radiolabel is not found associated with a distinctive cellular product. Similarly, ultrastructural studies of vertebrate steroid-synthesizing cells have not revealed any "recognizable accumulation of secretory material" (Fawcett et al., 1969). However, Scharrer (1971) reported cribriform bodies in the CA cells of L. maderae and, although they probably do not contain the JH itself, the author suggests that they may be involved, in some undetermined way, in the endocrine function of the CA. A biochemical study of these cribriform bodies would be useful to determine their role in JH biosynthesis and release.
Localization of Juvenile Hormone Biosynthesis
31
The close temporal relationship between hormone synthesis and release also suggests a close physical relationship between the site ofepoxidation and the site or release. Morphological evidence for this suggestion (Fig. 2) is a CA cell in which the SER (with grains) is located in close proximity to the extracellular compartment. Guelin and Darjo (1974) suggested the existence of three distinct regions in the typical CA cell of L. migratoria: the perinuclear area, the area of synthesis (secretion) and the area of interdigitation. The CA cells of S. gregaria appear to possess similar regions, although they are not as distinctly delineated. Grains were observed in all three regions, although seldom in the perinuclear area. SER and mitochondria are concentrated in the synthetic (secretory) region and much of the label is localized here. Grains can also be found in the region of interdigitation, associated with mitochondria and Golgi elements (Figs. 1, 4). These observations suggest that at least the final two stages in JH biosynthesis occur both in the region of synthesis and the region of interdigitation. However, each of the final enzymatic steps may be restricted to a particular region. For example, esterification may occur in the region of synthesis and epoxidation and release in the region of interdigitation. At present, it is not possible to localize separately the individual final stages. In conclusion, the present study clearly demonstrates that the major cellular organelles involved in the terminal stages of JH biosynthesis are the SER and possibly mitochondria and the Golgi complex. All cells of the CA of S. gregaria appear to possess the capacity to effect the final two stages in JH biosynthesis. Finally, the radiolabel cannot be associated with any distinctive cellular product. This is the first demonstration of the subcellular site of juvenile hormone biosynthesis in insects.
References Fain-Maurel, M.-A., Cassier, P.: Etude infrastructurale des corpora allata de Locusta migratoria migratoriodes (R. et F.), phase solitaire, au cours de la maturation sexuelle et des cycles ovariens. C.R. Acad. Sci. (Paris) 268 D, 2721-2723 (1969) Fawcett, D.A., Long, J~A., Jones, A.L.: The ultrastructure of endocrine glands. In: Recent Progr. Hormone Res. (E.B. Astwood, ed.) 25, 315-380 (1969) Guelin, M., Darjo, A.: Etude ultrastructurale des corpora allata en relation avec le contr61e photoperiodique de leur fonction gonadotrope chez Locusta migratoria migraWria L.C.R. Acad. Sci. (Paris) 278D, 491-494 (1974) Hammock, B.D.: NADPH dependent epoxidation of methyl farnesoate to juvenile hormone in the cockroach Blaberus giganteus L. Life Sci. 17, 323-328 (1975) Hirsch, J.G., Fedorko, M.E. : Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and postfixation in uranyl acetate. J. Cell Biol. 38, 615 627 (1968) Joly, L., Joly, P., Porte, A., Girardie, A.: Etude physiologique et ultrastructurale des corpora allata de Locusta migratoria L. (Orthopt~re) en phase gr6gaire. Arch. Zool. exp. g6n. 109, 703-728 (1968) Melnikova, E.J., Panov, A.A.: Ultrastructure of the larval corpus allatum of Hyphantria cunea Drury (Insecta, Lepidoptera). Cell Tiss. Res. 162, 395-410 (1975) Odhiambo, T.R.: The fine structure of the corpus allatum of the sexually mature male of the desert locust. J. Insect Physiol. 12, 819-828 (1966) Pratt, G.E., Tobe, S.S.: Juvenile hormones radiobiosynthesized by corpora allata of adult female locusts in vitro. Life Sci. 14, 575-586 (1974)
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Pratt, G.E., Tobe, S.S., Weaver, R.J.: Relative oxygenase activities in juvenile hormone biosynthesis of corpora allata of an African locust (Schistocerca gregaria) and American cockroach (Periplaneta americana). Experientia (Basel) 31, 120-122 (1975 b) Pratt, G.E., Tobe, S.S., Weaver, R.J., Finney, J.R.: Spontaneous synthesis and release of C16 juvenile hormone by isolated corpora allata of female locust Sehistocerca gregaria and female cockroach Periplaneta americana. Gen. comp. Endocr. 26, 478-484 (1975 a) Salpeter, M.M., Bachmann, L., Salpeter, E.E.: Resolution in electron microscope autoradiography. J. Cell Biol. 41, 1-20 (1969) Scharrer, B.: Histophysiological studies on the corpus aUatum of Leucophaea maderae. IV. Ultrastructure during normal activity cycle. Z. Zellforsch. 62, 125-148 (1964) Scharrer, B.: Histophysiological studies on the corpus allatum of Leucophaea maderae. V. Ultrastructure of sites of origin and release of a distinctive cellular product. Z. Zellforsch. 120, 1-16 (1971) Tobe, S.S., Pratt, G.E.: The influence of substrate concentrations on the rate of insect juvenile hormone biosynthesis by corpora allata of the desert locust in vitro. Biochem. J. 144, 107-113 (1974a) Tobe, S.S., Pratt, G.E.: Dependence of juvenile hormone release from corpus allatum on intraglandular content. Nature (Lond.) 252, 474-476 (1974 b) Tobe, S.S., Pratt, G.E.: Corpus allatum activity in vitro during ovarian maturation in the desert locust, Schistocerca gregaria. J. exp. Biol. 62, 611-627 (1975) Tobe, S.S., Pratt, G.E.: Farnesenic acid stimulation of juvenile hormone biosynthesis as an experimental probe in corpus allatum physiology. In: The juvenile hormones (L.I. Gilbert, ed.), pp. 147-163. New York: Plenum Press 1976 Tobe, S.S., Pratt, G.E., Saleuddin, A.S.M.: Intracellular visualization of C16 juvenile hormone biosynthesis in corpora allata of the adult female desert locust Schistocercagregaria. In: Actualit6s sur les Hormones d'Invert6br6s. Int. Colloq. C.N.R.S. No. 251, pp. 441-449, Paris (1976)
Accepted March 31, 1977
Cell and Tissue Research 9 by Springer-Verlag 1977
Ultrastructural Localization of Juvenile Hormone Biosynthesis by Insect Corpora Allata* S.S. Tobe Department of Zoology, University of Toronto, Toronto, Ontario, Canada A.S.M. Saleuddin Department of Biology, York University, Downsview, Ontario, Canada
Summary. The conversion of exogenous 3H-farnesenic acid to 3H-methyl farnesoate and 3H-C16 juvenile hormone (JH) has been followed in the corpus allatum (CA) cells of the desert locust Schistocerca gregaria by means of electron microscopic autoradiography. Aerobic and anaerobic 'chase' incubations have been used to modify the quantities of these three compounds within the CA cells. Under all incubation conditions, radiolabel is found associated almost exclusively with the subcellular m e m b r a n e s y s t e m s - smooth endoplasmic reticulum (SER) and Golgi elements - and with the mitochondria. CA cells are p r o b a b l y similar to vertebrate steroid-synthesizing cells in that the secretory product is synthesized in the SER and mitochondria. Radiolabel was found to be present in all cells of the CA suggesting that all cells are capable of at least the final two stages of J H biosynthesis (the esterification and epoxidation of 3H-farnesenic aid). This indicates that J H biosynthesis m a y be regulated through changes in the biosynthetic capabilities of individual cells rather than through changes in the total number of cells engaged in biosynthesis. Radiolabel was not observed to be associated with any distinctive cellular product, a result which provides additional evidence for the suggestion that the release of J H from the CA is governed by laws of simple physical diffusion. Key words: Corpus allatum - Juvenile hormone biosynthesis - Localization. Introduction The corpus allatum (CA) is the site of synthesis and release of the juvenile hormone (JH) in all insect species studied to date. In the desert locust, Schistocerca gregaria, the CA have been shown to synthesize and release C16JH (methyl 10,11-epoxySend offprint requests to: Dr. S.S. Tobe, Department of Zoology, University of Toronto, 25 Harbord St., Toronto, Ontario, Canada M5S 1A1
* Supported by operating grants from the National Research Council of Canada to SST and ASMS. 3H-farnesenic acid was supplied by the late Dr. A.F. White of the Unit of Invertebrate Chemistry and Physiology, A.R.C., University of Sussex. We thank Dr. G.E. Pratt for helpful discussions
26
S.S. Tobe and A.S.M. Saleuddin
3,7,11-trimethyl-2,6-trans, trans-dodecadienoate) when m a i n t a i n e d in vitro in a defined tissue culture m e d i u m for p e r i o d s o f u p to 5 h ( P r a t t a n d Tobe, 1974; T o b e a n d Pratt, 1974 a; P r a t t et al., 1975 a). T h u s the C A o f locusts have the c a p a b i l i t y to synthesize C16JH f r o m only simple p r e c u r s o r s present in the defined m e d i u m . In a d d i t i o n , locust C A can be s t i m u l a t e d to p r o d u c e larger quantities o f C a6JH by the a d d i t i o n o f the e x o g e n o u s p r e c u r s o r farnesenic acid to the i n c u b a t i o n m e d i u m ( P r a t t a n d Tobe, 1974; T o b e a n d Pratt, 1974a). It has been shown t h a t the e x o g e n o u s farnesenic acid is c o n v e r t e d to C16JH b y esterification a n d e p o x i d a t i o n a n d t h a t these final two stages in J H biosynthesis are n o t rate-limiting in C A o f S. gregaria ( P r a t t a n d Tobe, 1974; P r a t t et al., 1975 b). Because farnesenic acid is so readily i n c o r p o r a t e d into J H b y locust CA, we h y p o t h e s i z e d t h a t the r a d i o l a b e l l e d f o r m o f this c o m p o u n d c o u l d be used to ascertain the subcellular c o m p a r t m e n t s in which at least the final two stages o f J H biosynthesis occur. To this end, we have d e v e l o p e d a m e t h o d o l o g y which permits retention o f significant a m o u n t s o f r a d i o l a b e l within the C A after p r e p a r a t i o n for light a n d electron m i c r o s c o p i c a u t o r a d i o g r a p h y a n d following a variety o f pulse-chase p r o c e d u r e s ( T o b e a n d P r a t t , 1976; T o b e et al., 1976). In the p r e s e n t p a p e r we r e p o r t o n the s u b c e l l u l a r l o c a l i z a t i o n o f r a d i o l a b e l l e d p r o d u c t s as d e t e r m i n e d by electron m i c r o s c o p i c a u t o r a d i o g r a p h y . W e have r e p o r t e d previously on the light m i c r o s c o p i c localiz a t i o n o f r a d i o l a b e l l e d p r o d u c t s ( T o b e a n d Pratt, 1976). Materials and Methods
Schistocercagregariawere reared after the method of Tobe and Pratt (1975). Animals used in this study were females 2, 4 and 6 days after fledging (CA inactive) and 8 and 11 days after fledging (CA active). Corpora allata were dissected and incubated in the presence of 3H-farnesenic acid (final concentration 20 I~M)as described previously (Tobe and Pratt, 1976; Tobe et al., 1976). With radiochemical procedures, we have previously shown that under standard aerobic conditions, more than 50 % of the radiolabel within the CA is present as C 16JH after 30 min. When this standard incubation is followed by a twenty minute aerobic chase, more than 70 % of the radiolabel in the CA is present as C16JH, whereas if the standard incubation is followed by a twenty minute anaerobic chase (under nitrogen), approximately 50 % of the radiolabel is present as methyl farnesoate (Tobe and Pratt, 1976). We have employed these three treatments to obtain CA with varying amounts of radiolabelled farnesenic acid, methyl farnesoate and C16JH for evaluation by electron microscopic autoradiography. After the appropriate treatment, CA were fixed by a modification of the method of Hirsch and Fedorko (1968). The fixative consisted of a freshly prepared mixture of 2 parts 1% osmic acid in 0.1 M cacodylate buffer (pH 7.4) and 1 part 2.5 glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). The CA were embedded in Araldite 502 : Epon 812. For autoradiography, thin sections, placed on collodion-coated slides and allowed to dry, were stained with uranyl acetate and lead citrate, coated with a thin layer of carbon and dipped in Ilford L-4 emulsion after the method of Salpeter et al. (1969). Sections were incubated for 6-8 weeks, developed in Kodak D19 and examined in a Philips EM 201. Loss of label during each fixation and embedding step was monitored by counting aliquots of the fixative, ethanols and embedding media; this loss averaged 4050 % of the total label initially present.
Results and Discussion T h e C A o f S. gregaria are similar in m o r p h o l o g y to those o f o t h e r locust species ( G u e l i n a n d D a r j o , 1974). T h e y are c h a r a c t e r i z e d by an extensive s m o o t h e n d o p l a s m i c r e t i c u l u m (SER), n u m e r o u s m i t o c h o n d r i a a n d a p r o m i n e n t nucleus. In
Localizationof JuvenileHormoneBiosynthesis
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the CA cells ofS. gregaria, the mitochondria are large (Odhiambo, 1966), and small amounts of rough endoplasmic reticulum are present. In CA known to be synthesizing and releasing Ci6JH at a high rate (days 8-11 after fledging), numerous lysosomes, multivesicular bodies and Golgi elements also occur. Extensive extracellular stroma and axons, many containing neurosecretory granules, occur within the glands (Scharrer, 1964; Melnikova and Panov, 1975). Extracellular channels continuous with the gland's sheath afford communication with the haemocoel. The same system of extracellular channels carrying neurosecretory fibres occurs in the CA of Leucophaea maderae (Scharrer, 1964). The presence of abundant SER and mitochondria in CA cells has led many authors to suggest, by comparison with vertebrate steroid-synthesizing cells, that the synthetic product is lipoidal (see Fawcett et al., 1969). In S. gregaria the synthetic product is the sesquiterpenoid Ci6JH (Pratt and Tobe, 1974; Tobe and Pratt, 1974a, 1975) and, on this basis, it might be predicted that a significant proportion of the radiolabel would be associated with the SER and mitochondria. This is indeed the case; grains are localized almost exclusively on SER and occasional mitochondria and Golgi complexes (Figs. 1, 2). No difference in the distribution of radiolabel was observed between glands fixed immediately after 3H-farnesenic acid incubation and glands chased either aerobically or anaerobically before fixation. There is a difference in the total amount of label within the CA after these different treatments and this is reflected in the total number of grains observed by light microscopic autoradiography (Tobe and Pratt, 1976). Thus, although the total amounts of label observed within the CA after the three treatments may differ, the subcellular distribution of this label is similar in all cases. The use of these different treatments theoretically permitted the localization of specific metabolites in specific subcellular compartments, but since no differences in the distribution of label are apparent after these different treatments, we must conclude that the three metabolites which are being visualized by autoradiography are confined mainly to the SER and, to a lesser extent, to the mitochondria. Specific metabolites cannot be associated with specific organelles. Hammock (1975) reported that the final stage in JH biosynthesis, the epoxidation of methyl farnesoate, is largely confined to the microsomal fraction of the CA in Blaberus giganteus. This finding agrees well with the observed localization of radiolabel in the SER reported in the present paper. Two different types of SER can be observed in the CA cells of S. gregaria: concentric lamellar (Fig. 2) and vesiculate (Fig. 3). Label can be associated with both types. Both types have also been reported in the CA ofLocusta migratoria, although the functional difference between them remains unclear (Joly et al., 1968; FainMaurel and Cassier, 1969). These authors suggested that vesiculate SER is found in highly active glands whereas lamellar SER is found in inactive or involuting glands. Joly et al. (1968) further suggested that the fragmentation of the lamellar SER is important for the release of JH. However, our autoradiographic results do not support this contention since the label is not associated with any visible cellular product. The question of the number of cells actively engaged in the synthesis of JH has interested insect endocrinologists for some time. In a low magnification autoradiograph showing a number of different CA cells (Fig. 4), it can be seen that
Fig. 1. Autoradiograph of CA from 11 day S. gregaria showing grains on Golgi apparatus (arrow) and mitochondrion (white arrow). No chase. L lysosome, x 20,900 Fig. 2. Autoradiograph of CA from 11 day S. gregaria showing grains in concentric lamellae of smooth endoplasmic reticulum (SER). After 30min incubation in 3H-farnesenic acid, CA were chased aerobically for 20 min before fixation. T trachea. • 20,900
Localization of Juvenile Hormone Biosynthesis
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Fig 3. Autoradiograph of CA from 8 day S. gregaria showing grains on vesicular SER. No chase. L lysosome, x 20,900 Fig. 4. Autoradiograph of CA from 11 day S. gregaria showing random distribution of grains. No chase. Arrows indicate Golgi apparatus. L lysosome; M mitochondrion. • 15,200
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s.s. Tobe and A.S.M. Saleuddin
all cells contain grains. Therefore, all cells of the CA must be capable of at least esterification and epoxidation of 3H-farnesenic acid and this supports our previous light microscopic observation that all cells of the CA appear to be equally competent to effect the final two stages in JH biosynthesis (Tobe and Pratt, 1976). This result is significant in that the overall synthetic capacity of the CA is not regulated through the proportion of cells engaged in JH biosynthesis but rather through changes in the biosynthetic capabilities of individual cells. Guelin and Darjo (1974) reported that the CA cells of L. migratoria may function asynchronously and that thus not all cells may be equally capable of JH biosynthesis at any given time. It is conceivable that the secretory cells of the CA of S. gregaria undergo asynchronous cycles of JH biosynthesis but in the light of our radiochemical findings, which demonstrate large and predictable cyclical changes in JH biosynthesis in concert with the gonadotropic cycles, such asynchrony does not seem likely. Nonetheless, the methods used in the present study may not be sufficiently discriminating to permit the observation of different amounts of radiolabel within individual cells. Although total numbers of grains in individual cells were not determined, our previous light microscopic autoradiography indicated that the radiolabel is uniformly distributed throughout the glands, also suggesting that all CA cells are capable of effecting the final two stages in JH biosynthesis. It should be stressed that the use of 3H-farnesenic acid as a radiolabelled probe for the localization of JH biosynthesis reveals only the final two stages in the biosynthetic pathway. At present, the subcellular location of the early stages in JH biosynthesis is unknown although the synthesis of farnesyl pyrophosphate may follow the pathway described for vertebrate steroid-producing cells, i.e., SER and cytoplasmic matrix (Fawcett et al., 1969). An autoradiographic study of the early stages of JH biosynthesis may reveal areas of potential cytoplasmic compartmentalization which may be important in the regulation of overall JH biosynthesis by controlling the access of substrates to the appropriate enzyme compartments. As has been noted above, the radiolabel within the CA is present as 3H-Ca6JH and, to a lesser extent, as 3H-methyl farnesoate after different treatments. It is striking that the grains do not appear to be associated with any distinctive secretory product. It has been demonstrated that significant amounts of 3H-C16JH are present in the incubation medium within 10 min of the addition of 3H-farnesenic acid (Tobe and Pratt, 1974 a) and this observation led in part to the suggestion that the release of JH from the CA is governed by laws of simple physical diffusion; JH is released soon after it is synthesized (Tobe and Pratt, 1974 b). In view of the rapidity of release of the hormone, it is not surprising that the radiolabel is not found associated with a distinctive cellular product. Similarly, ultrastructural studies of vertebrate steroid-synthesizing cells have not revealed any "recognizable accumulation of secretory material" (Fawcett et al., 1969). However, Scharrer (1971) reported cribriform bodies in the CA cells of L. maderae and, although they probably do not contain the JH itself, the author suggests that they may be involved, in some undetermined way, in the endocrine function of the CA. A biochemical study of these cribriform bodies would be useful to determine their role in JH biosynthesis and release.
Localization of Juvenile Hormone Biosynthesis
31
The close temporal relationship between hormone synthesis and release also suggests a close physical relationship between the site ofepoxidation and the site or release. Morphological evidence for this suggestion (Fig. 2) is a CA cell in which the SER (with grains) is located in close proximity to the extracellular compartment. Guelin and Darjo (1974) suggested the existence of three distinct regions in the typical CA cell of L. migratoria: the perinuclear area, the area of synthesis (secretion) and the area of interdigitation. The CA cells of S. gregaria appear to possess similar regions, although they are not as distinctly delineated. Grains were observed in all three regions, although seldom in the perinuclear area. SER and mitochondria are concentrated in the synthetic (secretory) region and much of the label is localized here. Grains can also be found in the region of interdigitation, associated with mitochondria and Golgi elements (Figs. 1, 4). These observations suggest that at least the final two stages in JH biosynthesis occur both in the region of synthesis and the region of interdigitation. However, each of the final enzymatic steps may be restricted to a particular region. For example, esterification may occur in the region of synthesis and epoxidation and release in the region of interdigitation. At present, it is not possible to localize separately the individual final stages. In conclusion, the present study clearly demonstrates that the major cellular organelles involved in the terminal stages of JH biosynthesis are the SER and possibly mitochondria and the Golgi complex. All cells of the CA of S. gregaria appear to possess the capacity to effect the final two stages in JH biosynthesis. Finally, the radiolabel cannot be associated with any distinctive cellular product. This is the first demonstration of the subcellular site of juvenile hormone biosynthesis in insects.
References Fain-Maurel, M.-A., Cassier, P.: Etude infrastructurale des corpora allata de Locusta migratoria migratoriodes (R. et F.), phase solitaire, au cours de la maturation sexuelle et des cycles ovariens. C.R. Acad. Sci. (Paris) 268 D, 2721-2723 (1969) Fawcett, D.A., Long, J~A., Jones, A.L.: The ultrastructure of endocrine glands. In: Recent Progr. Hormone Res. (E.B. Astwood, ed.) 25, 315-380 (1969) Guelin, M., Darjo, A.: Etude ultrastructurale des corpora allata en relation avec le contr61e photoperiodique de leur fonction gonadotrope chez Locusta migratoria migraWria L.C.R. Acad. Sci. (Paris) 278D, 491-494 (1974) Hammock, B.D.: NADPH dependent epoxidation of methyl farnesoate to juvenile hormone in the cockroach Blaberus giganteus L. Life Sci. 17, 323-328 (1975) Hirsch, J.G., Fedorko, M.E. : Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and postfixation in uranyl acetate. J. Cell Biol. 38, 615 627 (1968) Joly, L., Joly, P., Porte, A., Girardie, A.: Etude physiologique et ultrastructurale des corpora allata de Locusta migratoria L. (Orthopt~re) en phase gr6gaire. Arch. Zool. exp. g6n. 109, 703-728 (1968) Melnikova, E.J., Panov, A.A.: Ultrastructure of the larval corpus allatum of Hyphantria cunea Drury (Insecta, Lepidoptera). Cell Tiss. Res. 162, 395-410 (1975) Odhiambo, T.R.: The fine structure of the corpus allatum of the sexually mature male of the desert locust. J. Insect Physiol. 12, 819-828 (1966) Pratt, G.E., Tobe, S.S.: Juvenile hormones radiobiosynthesized by corpora allata of adult female locusts in vitro. Life Sci. 14, 575-586 (1974)
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Pratt, G.E., Tobe, S.S., Weaver, R.J.: Relative oxygenase activities in juvenile hormone biosynthesis of corpora allata of an African locust (Schistocerca gregaria) and American cockroach (Periplaneta americana). Experientia (Basel) 31, 120-122 (1975 b) Pratt, G.E., Tobe, S.S., Weaver, R.J., Finney, J.R.: Spontaneous synthesis and release of C16 juvenile hormone by isolated corpora allata of female locust Sehistocerca gregaria and female cockroach Periplaneta americana. Gen. comp. Endocr. 26, 478-484 (1975 a) Salpeter, M.M., Bachmann, L., Salpeter, E.E.: Resolution in electron microscope autoradiography. J. Cell Biol. 41, 1-20 (1969) Scharrer, B.: Histophysiological studies on the corpus aUatum of Leucophaea maderae. IV. Ultrastructure during normal activity cycle. Z. Zellforsch. 62, 125-148 (1964) Scharrer, B.: Histophysiological studies on the corpus allatum of Leucophaea maderae. V. Ultrastructure of sites of origin and release of a distinctive cellular product. Z. Zellforsch. 120, 1-16 (1971) Tobe, S.S., Pratt, G.E.: The influence of substrate concentrations on the rate of insect juvenile hormone biosynthesis by corpora allata of the desert locust in vitro. Biochem. J. 144, 107-113 (1974a) Tobe, S.S., Pratt, G.E.: Dependence of juvenile hormone release from corpus allatum on intraglandular content. Nature (Lond.) 252, 474-476 (1974 b) Tobe, S.S., Pratt, G.E.: Corpus allatum activity in vitro during ovarian maturation in the desert locust, Schistocerca gregaria. J. exp. Biol. 62, 611-627 (1975) Tobe, S.S., Pratt, G.E.: Farnesenic acid stimulation of juvenile hormone biosynthesis as an experimental probe in corpus allatum physiology. In: The juvenile hormones (L.I. Gilbert, ed.), pp. 147-163. New York: Plenum Press 1976 Tobe, S.S., Pratt, G.E., Saleuddin, A.S.M.: Intracellular visualization of C16 juvenile hormone biosynthesis in corpora allata of the adult female desert locust Schistocercagregaria. In: Actualit6s sur les Hormones d'Invert6br6s. Int. Colloq. C.N.R.S. No. 251, pp. 441-449, Paris (1976)
Accepted March 31, 1977
