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Stimulatory activity of cysteamine on juvenile hormone release in adult females of the cockroach, Blattella germanica
Camp. Biodwm. Ph.wio/. Printed in Great Bntain
0300-9629,‘89 $3.00+ 0.00 c 1989Pergamon Press plc
Vol. 94A, No. 4. pp. 795-798, 1989
STIMULATORY ACTIVITY OF CYSTEAMINE ON JUVENILE HORMONE RELEASE IN ADULT FEMALES THE COCKROACH, BLATTELLA GERMANICA
OF
MARIA-DOLORS PIULACHS and XAVIER BELL&* Department of Biological Organic Chemistry (C.I.D., C.S.I.C.) Jordi Girona, 18. 08034-Barcelona, Spain. Telephone: 204-0600 (Receioed 14 M~J? 1989) Abstract-l. A study of the activity of cysteamme in relation to juvenile hormone (JH) production in adult females of BlulrrUu germanica was carried out. 2. In vizw assays showed that cysteamine stimulates protein synthesis in the left colleterial gland and, in some instances, enhances oocyte growth. 3. In oifro assays demonstrated that cysteamine enhances JH release by incubated corpora allata (CA), and that this effect is more pronounced when using CA from IO-day-old females (period of ootheca transport). either connected to the corpora cardiaca (CC) or to the CC and to the brain. 4. Possible antiallatostatic effects of cysteamine are discussed.
Compounds
INTRODUCTION
Cysteamine hydrochloride (Merck) was dissolved in sterile TC-199 medium (see below) at appropriate concentrations, either for in oiuo or in uifro assays. Juvenile hormone III was obtained from Sigma. [IO-‘H(N)]-juvenile hormone III (11 Ci/mmol) was from New England Nuclear, and [methyl-‘Hlmethionine (SO-85 Ci/mmol) from Amersham. Solvents were all of analytical grade and used as received.
Cysteamine (mercaptoethylamine, HS-CH,-CH2NH,) is a thiol reagent which elicits a number of biological activities in mammals. Pharmacologically, this compound behaves as a dopamine-beta-hydroxylase inhibitor, thus influencing the metabolism of catecholamines. In rats it depletes immunoreactive somatostatin, gonadotropins, thyrotropic hormone, prolactin and beta-endorphin-like immunoreactivity. In addition, cysteamine disrupts growth hormone release by affecting both the somatostatin inhibiting and growth-hormone-releasing-factor stimulating components (see Webb ef al., 1986, and enclosed references). In a previous paper on the inhibitory activity of somatostatin on juvenile hormone (JH) release in the cockroach Blattella germanica (Bell&s and Piulachs, 1988), we included preliminary data on the inverse activity induced by cysteamine. Here, we present our results of further research in this line, by studying the effects of cysteamine on different developmental stages in the first gonotropic cycle of this cockroach. Experiments were conducted either in oiro, to study the activity of cysteamine on oocyte growth and on left colleterial gland development, or in L?tro, investigating its influence on JH release by incubated corpora allata. MATERIALS AND
In vivo assays Cysteamine was injected at appropriate concentrations in a volume of 2~1 in the penultimate intersegmental membrane of the sternal part of the abdomen. Controls received the same volume of TC-199 medium. Assays were made on virgin females, either freshly ecdysed or IO-day-old. i.e. in the period of ootheca transport, three days after oviposition. Oocyte growth was checked I day after the treatment in the case of freshly ecdysed females, or 4 days after the treatment in those experiments performed with IO-day-old females. Quantification of oocyte growth was carried out by measuring and averaging three basal oocytes of each ovary per specimen, and that of colleterial gland development was calculated by measuring and averaging the diameter of five tubules selected at random of the left colleterial gland. The presence of protein in this gland was estimated considering the following three categories: (0) empty glands, showing a transparent aspect; (I) partially filled glands, showing a thin filament of milky white secretion along each tubule; and (2) filled and opaque glands. each tubule showing a uniform milky white colour. The three categories are found in the same interval of diameter of the tubules (5-9 x IO-*mm) corresponding to pre- or post-vitellogenic females (see Bell&s and Piulachs, 1983).
METHODS
Insects
In vitro ussu)rs
Specimens of Bluttellu germunicu were obtained from a colony reared, at 30( k l).C in complete darkness, with dog food and water ad libitum. Freshly ecdysed adult females were isolated and maintained in the absence of males in the same environmental conditions. and were used at appropriate ages, as indicated.
*Author
to whom
all correspondence
should
Dissection of glands (connected brainxorpora cardiacacorpora allata: BZC-CA; connected corpora cardiacacorpora allata: CCCA; or isolated corpora allata: CA) were carried out as previously described (Bell&s and Piulachs, in press). Dissected glands were transferred to 230 p I of Millipore filtered TC-199 medium containing L-methionine (0.05 mM). Hank’s salts, Hepes buffer (20 mM) plus Ficoll (20 mg/ml) and to which L-(methyl-‘H)methionine (diluted to a final specific activity of 400 mCi!mmol) was
be addressed. 795
796
MARIA-DOLORS PIULACHS and XAVIER BELLES
added. Rates of JH release were determined by the radiochemical method of Tobe and Pratt (1974), which was further adapted to the case of B. germanica by Belles et al. (1987). The experimental design comprises an incubation period of 2 hr in TC-199 fresh medium, followed by transference of the glands to a medium containing the desired concentration of cysteamine, when the incubation is pursued for 2 hr. Glands are then incubated again in fresh medium for 2 hr to study the persistence of the effects observed in the preceding period.
RESULTS
In vivo experiments In B. ger~un~c~, either
ovarian
as it is the case in many
growth
as well as protein
insects, synthesis in
left coileterial gland are both dependent on the release of JH by the CA (see Koeppe et al., 1985, for review). Therefore, there is a cycle of JH release which parallels the development of both organs. After oviposition, and during the period of ootheca transport, the CA release low amounts of JH, as in freshly ecdysed specimens, and basal oocyte growth and left colleterial gland development are arrested until the nymphs hatch (Roth and Stay, 1962; BeEs and Piulachs, 1983; Belles et a/., 1987). The aim of the in uivo experiments was to study if cysteamine could stimulate basal oocyte growth and left colleterial gland development in inactive females, thus providing an indirect indication of CA stimulation. Therefore, two different ages where chosen for these purposes: freshly ecdysed (O-.24 hr after the imaginal ecdysis) and lo-day-old (period of oothecd transport, 3 days after oviposition). The results obtained (Table 1) show that cysteamine only induced a significant stimulation of oocyte growth and colleterial gland development in the case of IOpg treatment on freshly ecdysed females (t-test. P < 0.05). In addition, this was the sole experiment in which the compound induced toxic effects, resulting in a 67% of mortality. Values of the length of basal oocyte, and of the diameter of the left colleterial gland tubules obtained from the other treatments depicted in Table 1, showed no significant differences in comparison with their respective controls. However, data on the presence of milky white secretion in the tubules of the left colleterial gland (see Table l), were apparent enough to suggest that cysteamine treatment resulted in an enhancement of
protein synthesis in such a gland, either ecdysed as well as in IO-day-old females.
in freshly
In vitro experiments In order to confirm a possible stimulatory activity of cysteamine on JH release, a series of in vitro experiments were carried out by incubating the CA in the presence of the compound. Therefore, the activity of cysteamine was first studied on incubations of the whole retrocerebral complex (CC-CA) from virgin females representing three characteristic developmental stages in the first gonotropic cycle: (a) freshly ecdysed (previtellogenesis). (b) 6-day-old (vitellogenesis), and (c) IO-day-old (postvitellogenesis). The results of these experiments (Fig. I) showed that JH release was significantly enhanced by cysteamine in glands from freshly ecdysed (at IO-’ M) or from IO-day-old females (at 10 ,3 and 10m4 M). In this last group, cysteamine was ineffective when the dose was lowered to lO-‘M (results not shown). In a second set of experiments, the activity of cysteamine on in vitro JH release was additionally studied in glands from IO-day-old females, either incubating the CA alone. or the CA connected to the CC and to the brain (B-CC--CA). As summarized in Fig. 1. the concentration of IO- ‘M significantly enhanced JH release in both cases, just as it did in CC-CA incubations (see above). However, when the dose was lowered to 10 4 M, the CA incubated alone were no longer stimulated by the compound. whereas in R-CC-CA incubations JH release was still enhanced. As in the case of CC--CA, the dose of IO 5hl also resulted inactive in B-CC CA experiments (results not shown). IXSCUSSION
The symptoms of protein synthesis activation in the left colleterial gland in inactive females of B. germanica, treated with cysteamine. provided an indirect indication accounting for a possible stimulatory activity of this compound on JH production. This was further confirmed by more specific in vitro assays incubating the CA in the presence of cysteamine. Results of the in vitro experiments comparing three different ages of gland donors: freshly ecdysed. &day-old, and IO-day-old, showed that the stimulatory effects of cysteamine on JH release were more pronounced on glands from IO-day-old females.
Table I. Effects of cysteamine (cys) on oocyte growth and left colletetial gland development in virgin females of SIuririfu gemranin~ Experiments were carried out on freshly ecdysed (FE) and on IO-day-old (IOD) specimens. and the effects were checked 1 or 4 days after the treatment, respectively Specmvxs showmg d!@xent levels of secretion in the left collaterial glandt Age ~_ FE FE FE 10D IOD 10D 1OD
Treatment ._.___~~--.. 1oPg CYS Ipgcys (control) lO/Lgcys lpgcys 0.t fig cys (control)
Basal oocyte N~-..- lengh (mm)* 6 IO 10 14 10 6
1s
0.74 0.53 0.53 0.39 0.38
Colleterial
gland
x IO ’ )* ~___._~~ _-diameter -. (mm
0
I
I I
2 4 1
& 0.12 i 0.01 i 0.01 1_ 0.01 F 0.01
8.582 I.16 6.28 * 0.50 6.45 f 0.39 5.69 i 0.32 5.68 IO.29
5 4 0 2
4 5 n
I4
0.45 & 0.03 0.40 i 0.01
5.20 + 0.31 5.16 & 0.21
4 II
1 2
1 2
1
*Mean i SEM. tResponse categories: (0) empty and transparent glands; (I) glands showing a thin filament of white secretion tubule; (2) glands showing a uniform milky white colour in each tubule (see Materials and Methods)
7
along rach
197
Cysteamine enhances JH release
k
Freshly
2 _
ecdysed
females
IO-Day-old
6-Day-old
females
females
0)
1 c
T IO
3
05
Brain-corpora
cardiaca-corpora allata
Corpora
0
Pre-treatment
m
Treatment
~
Post-treatment
(O-2hr)
(2-4hr) (4-6hr)
allata
Fig. 1. In cirro effects of cysteamine on juvenile hormone release @mol/hr per pair CA) by corpora allata from virgin females of Blattella germanica of different ages (corpora cardiacaxorpora allata incubations, upper histograms) and using different gland combinations (lo-day-old females, lower histograms). The number of individual determinations is indicated at the top of each experiment, and vertical bars represent the standard error of the mean. Results of f-test for paired data (from the comparison of the values resulting in the period of treatment with those obtained in the pre-treatment period of the same experiment) are summarized as follows: **(P < O.OOl), ‘(P < 0.01). Interestingly, these females, which are in the period of ootheca transport, show a low spontaneous production of JH, which is apparently due to the action of endogenous allatostatic factors (see below). Moreover, the in vitro studies carried out on IOday-old females on the influence of innervation from the brain to the CA, evidenced that the stimulatory action of cysteamine is more effective when the CA are either connected to the CC or to the CC and to the brain. Considering the well-known antagonistic action of cysteamine on vertebrate regulatory peptides (see Webb et al., 1986) and the whole results herein reported, the hypothesis that could be postulated to explain the stimulatory activity of this compound on JH production is an antagonistic action on the allatostatic factors present in the brain and in the CC. In fact, allatostatic activity has been recently found in brain extracts of I O-day-old females of B. german ica, and nervoctomy experiments in this species have evidenced that the factors eliciting this activity are transported to the CC and to the CA through the nervous connexions (Bell& and Piulachs, in press). Allatostatic factors have also been documented in other cockroaches, for example in Periplaneta americana (Pipa, 1982), although the species which has been more thoroughly studied in this sense is Diploptera punctata, in which the occurrence of peptidic allatostatic factors has been demonstrated in different tissues (see Rankin and Stay, 1987; Woodhead et al., 1989). Indiscriminate stimulatory activity of cysteamine in all experiments applying a dose of 10d3 M could be explained by an unspecific action of this compound on JH biosynthesis. The reported effects of cysteamine on dopamine accumulation in vertebrate nervous tissues (Webb et al., 1986), could afford a possible explanation in this sense, since this biogenic
amine has been detected in the CA of cockroaches and seems to have a beneficial action on JH production (see Hentschel, 1981). Although the mechanisms by which cysteamine exerts its stimulatory activity on JH release cannot be elucidated with the data presently available, we think that this compound could be a useful tool to investigate the syndromes of overproduction of JH in sensitive insects. Furthermore, and to the best of our knowledge, this is the first time that an antagonist of vertebrate peptidic hormones shows a biological activity, which could be analogous, in insects. This provides further support to the functional analogies of vertebrate and invertebrate endocrine systems postulated by different authors, and underlines once again the suitability of insects as models for neuroendocrine research (see Scharrer, 1987). Acknow[edRements-Thanks are due to Dr Francisco Camps for critical reading of the manuscript. Funds from CAICYT (project 84-0087) and CSIC (project 263-85) are also gratefully acknowledged.
REFERENCES
Bell&s X. and Piulachs M. D. (1983) Desarrollo de 10s corpora allata, oocitos y glindulas colaterales durante el primer ciclo gonotr6fico de Blattella germanica. Rev. Esp. Fisid. 39, 149-154. Bell&s X. and Piulachs M. D. (1988) Modulation by somatostatin of juvenile hormone release in a cockroach. Nuturwissenschaften 15, 413415. Bell&s X. and Piulachs M. D. (1989) Occurrence of brain allatostatic factors in the cockroach BIaffella germanica. Acta Entomol. Bohemoslov. 86 (in press). Bell&s X., Casas J., Messeguer A. and Piulachs M. D. (1987) In vitro biosynthesis of JH III by the corpora allata of adult females of Biattella germanica (L). Insect Biochem. 17, 1007-1010.
798
MARIA-DOLORS PIULACHS and XAVIER BELLES
Hentschel E. (1981) Investigations on catecholamine integrated influences on reproduction in Periplanera americana (L.). Adv. Physiol.. Sri. 22, 205-230.’ Koenoe J. K.. Fuchs M.. Chen T. T.. Hunt L. M.. Kovalich G: ‘E. and Briers T. (1985) The role of juvenile hormone in reproduction. In Comprehensice Insecr Physiology. Biochemistry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. T.), Vol. 8, pp. 1655204. Pergamon Press, Oxford. Pipa R. L. (1982) Neural influence on corpus allatum activity and egg maturation in starved virgin Periplanera americana. Physiol. Entomol. 7, 449455. Rankin S. M. and Stay B. (1987) Distribution of allatostatin in the adult cockroach, Diplopterapuncrata and effects on corpora allata in aifro. J. Insecf Physiol. 33, 551-558. Roth L. M. and Stay B. (1962) Oocyte development in
Blattella germanica and Blartella c’aga (Blattaria). A. MI. Sot. Am. 55, 633-642. Scharrer B. (1987) Insects as models in neuroendocrine research. Ann. Rec. Enromol. 32, I-16. Tobe S. S. and Pratt G. E. (1974) The influence of substrate concentration on the rate of insect juvenile hormone biosynthesis by corpora allata of the desert locust in vitro Biochem. J. 144, 107-l 13. Webb S. M., Champney T. H., Steger R. W.. Vaughan M. K. and Reiter R. J. (1986) Cysteamine effects on somatostatin, catecholamines, pineal NAT and melatonin in rats. Brain Res. Bull. 16, 315 320. Woodhead A. P., Stay B., Seidel S. L., Khan M. A. and Tobe S. S. (1989) Primary structure of four allatostatim neuropeptide inhibitors of juvenile hormone synthesis. Proc. natn. Acad. Sri. USA 86, 5997-6001.
0300-9629,‘89 $3.00+ 0.00 c 1989Pergamon Press plc
Vol. 94A, No. 4. pp. 795-798, 1989
STIMULATORY ACTIVITY OF CYSTEAMINE ON JUVENILE HORMONE RELEASE IN ADULT FEMALES THE COCKROACH, BLATTELLA GERMANICA
OF
MARIA-DOLORS PIULACHS and XAVIER BELL&* Department of Biological Organic Chemistry (C.I.D., C.S.I.C.) Jordi Girona, 18. 08034-Barcelona, Spain. Telephone: 204-0600 (Receioed 14 M~J? 1989) Abstract-l. A study of the activity of cysteamme in relation to juvenile hormone (JH) production in adult females of BlulrrUu germanica was carried out. 2. In vizw assays showed that cysteamine stimulates protein synthesis in the left colleterial gland and, in some instances, enhances oocyte growth. 3. In oifro assays demonstrated that cysteamine enhances JH release by incubated corpora allata (CA), and that this effect is more pronounced when using CA from IO-day-old females (period of ootheca transport). either connected to the corpora cardiaca (CC) or to the CC and to the brain. 4. Possible antiallatostatic effects of cysteamine are discussed.
Compounds
INTRODUCTION
Cysteamine hydrochloride (Merck) was dissolved in sterile TC-199 medium (see below) at appropriate concentrations, either for in oiuo or in uifro assays. Juvenile hormone III was obtained from Sigma. [IO-‘H(N)]-juvenile hormone III (11 Ci/mmol) was from New England Nuclear, and [methyl-‘Hlmethionine (SO-85 Ci/mmol) from Amersham. Solvents were all of analytical grade and used as received.
Cysteamine (mercaptoethylamine, HS-CH,-CH2NH,) is a thiol reagent which elicits a number of biological activities in mammals. Pharmacologically, this compound behaves as a dopamine-beta-hydroxylase inhibitor, thus influencing the metabolism of catecholamines. In rats it depletes immunoreactive somatostatin, gonadotropins, thyrotropic hormone, prolactin and beta-endorphin-like immunoreactivity. In addition, cysteamine disrupts growth hormone release by affecting both the somatostatin inhibiting and growth-hormone-releasing-factor stimulating components (see Webb ef al., 1986, and enclosed references). In a previous paper on the inhibitory activity of somatostatin on juvenile hormone (JH) release in the cockroach Blattella germanica (Bell&s and Piulachs, 1988), we included preliminary data on the inverse activity induced by cysteamine. Here, we present our results of further research in this line, by studying the effects of cysteamine on different developmental stages in the first gonotropic cycle of this cockroach. Experiments were conducted either in oiro, to study the activity of cysteamine on oocyte growth and on left colleterial gland development, or in L?tro, investigating its influence on JH release by incubated corpora allata. MATERIALS AND
In vivo assays Cysteamine was injected at appropriate concentrations in a volume of 2~1 in the penultimate intersegmental membrane of the sternal part of the abdomen. Controls received the same volume of TC-199 medium. Assays were made on virgin females, either freshly ecdysed or IO-day-old. i.e. in the period of ootheca transport, three days after oviposition. Oocyte growth was checked I day after the treatment in the case of freshly ecdysed females, or 4 days after the treatment in those experiments performed with IO-day-old females. Quantification of oocyte growth was carried out by measuring and averaging three basal oocytes of each ovary per specimen, and that of colleterial gland development was calculated by measuring and averaging the diameter of five tubules selected at random of the left colleterial gland. The presence of protein in this gland was estimated considering the following three categories: (0) empty glands, showing a transparent aspect; (I) partially filled glands, showing a thin filament of milky white secretion along each tubule; and (2) filled and opaque glands. each tubule showing a uniform milky white colour. The three categories are found in the same interval of diameter of the tubules (5-9 x IO-*mm) corresponding to pre- or post-vitellogenic females (see Bell&s and Piulachs, 1983).
METHODS
Insects
In vitro ussu)rs
Specimens of Bluttellu germunicu were obtained from a colony reared, at 30( k l).C in complete darkness, with dog food and water ad libitum. Freshly ecdysed adult females were isolated and maintained in the absence of males in the same environmental conditions. and were used at appropriate ages, as indicated.
*Author
to whom
all correspondence
should
Dissection of glands (connected brainxorpora cardiacacorpora allata: BZC-CA; connected corpora cardiacacorpora allata: CCCA; or isolated corpora allata: CA) were carried out as previously described (Bell&s and Piulachs, in press). Dissected glands were transferred to 230 p I of Millipore filtered TC-199 medium containing L-methionine (0.05 mM). Hank’s salts, Hepes buffer (20 mM) plus Ficoll (20 mg/ml) and to which L-(methyl-‘H)methionine (diluted to a final specific activity of 400 mCi!mmol) was
be addressed. 795
796
MARIA-DOLORS PIULACHS and XAVIER BELLES
added. Rates of JH release were determined by the radiochemical method of Tobe and Pratt (1974), which was further adapted to the case of B. germanica by Belles et al. (1987). The experimental design comprises an incubation period of 2 hr in TC-199 fresh medium, followed by transference of the glands to a medium containing the desired concentration of cysteamine, when the incubation is pursued for 2 hr. Glands are then incubated again in fresh medium for 2 hr to study the persistence of the effects observed in the preceding period.
RESULTS
In vivo experiments In B. ger~un~c~, either
ovarian
as it is the case in many
growth
as well as protein
insects, synthesis in
left coileterial gland are both dependent on the release of JH by the CA (see Koeppe et al., 1985, for review). Therefore, there is a cycle of JH release which parallels the development of both organs. After oviposition, and during the period of ootheca transport, the CA release low amounts of JH, as in freshly ecdysed specimens, and basal oocyte growth and left colleterial gland development are arrested until the nymphs hatch (Roth and Stay, 1962; BeEs and Piulachs, 1983; Belles et a/., 1987). The aim of the in uivo experiments was to study if cysteamine could stimulate basal oocyte growth and left colleterial gland development in inactive females, thus providing an indirect indication of CA stimulation. Therefore, two different ages where chosen for these purposes: freshly ecdysed (O-.24 hr after the imaginal ecdysis) and lo-day-old (period of oothecd transport, 3 days after oviposition). The results obtained (Table 1) show that cysteamine only induced a significant stimulation of oocyte growth and colleterial gland development in the case of IOpg treatment on freshly ecdysed females (t-test. P < 0.05). In addition, this was the sole experiment in which the compound induced toxic effects, resulting in a 67% of mortality. Values of the length of basal oocyte, and of the diameter of the left colleterial gland tubules obtained from the other treatments depicted in Table 1, showed no significant differences in comparison with their respective controls. However, data on the presence of milky white secretion in the tubules of the left colleterial gland (see Table l), were apparent enough to suggest that cysteamine treatment resulted in an enhancement of
protein synthesis in such a gland, either ecdysed as well as in IO-day-old females.
in freshly
In vitro experiments In order to confirm a possible stimulatory activity of cysteamine on JH release, a series of in vitro experiments were carried out by incubating the CA in the presence of the compound. Therefore, the activity of cysteamine was first studied on incubations of the whole retrocerebral complex (CC-CA) from virgin females representing three characteristic developmental stages in the first gonotropic cycle: (a) freshly ecdysed (previtellogenesis). (b) 6-day-old (vitellogenesis), and (c) IO-day-old (postvitellogenesis). The results of these experiments (Fig. I) showed that JH release was significantly enhanced by cysteamine in glands from freshly ecdysed (at IO-’ M) or from IO-day-old females (at 10 ,3 and 10m4 M). In this last group, cysteamine was ineffective when the dose was lowered to lO-‘M (results not shown). In a second set of experiments, the activity of cysteamine on in vitro JH release was additionally studied in glands from IO-day-old females, either incubating the CA alone. or the CA connected to the CC and to the brain (B-CC--CA). As summarized in Fig. 1. the concentration of IO- ‘M significantly enhanced JH release in both cases, just as it did in CC-CA incubations (see above). However, when the dose was lowered to 10 4 M, the CA incubated alone were no longer stimulated by the compound. whereas in R-CC-CA incubations JH release was still enhanced. As in the case of CC--CA, the dose of IO 5hl also resulted inactive in B-CC CA experiments (results not shown). IXSCUSSION
The symptoms of protein synthesis activation in the left colleterial gland in inactive females of B. germanica, treated with cysteamine. provided an indirect indication accounting for a possible stimulatory activity of this compound on JH production. This was further confirmed by more specific in vitro assays incubating the CA in the presence of cysteamine. Results of the in vitro experiments comparing three different ages of gland donors: freshly ecdysed. &day-old, and IO-day-old, showed that the stimulatory effects of cysteamine on JH release were more pronounced on glands from IO-day-old females.
Table I. Effects of cysteamine (cys) on oocyte growth and left colletetial gland development in virgin females of SIuririfu gemranin~ Experiments were carried out on freshly ecdysed (FE) and on IO-day-old (IOD) specimens. and the effects were checked 1 or 4 days after the treatment, respectively Specmvxs showmg d!@xent levels of secretion in the left collaterial glandt Age ~_ FE FE FE 10D IOD 10D 1OD
Treatment ._.___~~--.. 1oPg CYS Ipgcys (control) lO/Lgcys lpgcys 0.t fig cys (control)
Basal oocyte N~-..- lengh (mm)* 6 IO 10 14 10 6
1s
0.74 0.53 0.53 0.39 0.38
Colleterial
gland
x IO ’ )* ~___._~~ _-diameter -. (mm
0
I
I I
2 4 1
& 0.12 i 0.01 i 0.01 1_ 0.01 F 0.01
8.582 I.16 6.28 * 0.50 6.45 f 0.39 5.69 i 0.32 5.68 IO.29
5 4 0 2
4 5 n
I4
0.45 & 0.03 0.40 i 0.01
5.20 + 0.31 5.16 & 0.21
4 II
1 2
1 2
1
*Mean i SEM. tResponse categories: (0) empty and transparent glands; (I) glands showing a thin filament of white secretion tubule; (2) glands showing a uniform milky white colour in each tubule (see Materials and Methods)
7
along rach
197
Cysteamine enhances JH release
k
Freshly
2 _
ecdysed
females
IO-Day-old
6-Day-old
females
females
0)
1 c
T IO
3
05
Brain-corpora
cardiaca-corpora allata
Corpora
0
Pre-treatment
m
Treatment
~
Post-treatment
(O-2hr)
(2-4hr) (4-6hr)
allata
Fig. 1. In cirro effects of cysteamine on juvenile hormone release @mol/hr per pair CA) by corpora allata from virgin females of Blattella germanica of different ages (corpora cardiacaxorpora allata incubations, upper histograms) and using different gland combinations (lo-day-old females, lower histograms). The number of individual determinations is indicated at the top of each experiment, and vertical bars represent the standard error of the mean. Results of f-test for paired data (from the comparison of the values resulting in the period of treatment with those obtained in the pre-treatment period of the same experiment) are summarized as follows: **(P < O.OOl), ‘(P < 0.01). Interestingly, these females, which are in the period of ootheca transport, show a low spontaneous production of JH, which is apparently due to the action of endogenous allatostatic factors (see below). Moreover, the in vitro studies carried out on IOday-old females on the influence of innervation from the brain to the CA, evidenced that the stimulatory action of cysteamine is more effective when the CA are either connected to the CC or to the CC and to the brain. Considering the well-known antagonistic action of cysteamine on vertebrate regulatory peptides (see Webb et al., 1986) and the whole results herein reported, the hypothesis that could be postulated to explain the stimulatory activity of this compound on JH production is an antagonistic action on the allatostatic factors present in the brain and in the CC. In fact, allatostatic activity has been recently found in brain extracts of I O-day-old females of B. german ica, and nervoctomy experiments in this species have evidenced that the factors eliciting this activity are transported to the CC and to the CA through the nervous connexions (Bell& and Piulachs, in press). Allatostatic factors have also been documented in other cockroaches, for example in Periplaneta americana (Pipa, 1982), although the species which has been more thoroughly studied in this sense is Diploptera punctata, in which the occurrence of peptidic allatostatic factors has been demonstrated in different tissues (see Rankin and Stay, 1987; Woodhead et al., 1989). Indiscriminate stimulatory activity of cysteamine in all experiments applying a dose of 10d3 M could be explained by an unspecific action of this compound on JH biosynthesis. The reported effects of cysteamine on dopamine accumulation in vertebrate nervous tissues (Webb et al., 1986), could afford a possible explanation in this sense, since this biogenic
amine has been detected in the CA of cockroaches and seems to have a beneficial action on JH production (see Hentschel, 1981). Although the mechanisms by which cysteamine exerts its stimulatory activity on JH release cannot be elucidated with the data presently available, we think that this compound could be a useful tool to investigate the syndromes of overproduction of JH in sensitive insects. Furthermore, and to the best of our knowledge, this is the first time that an antagonist of vertebrate peptidic hormones shows a biological activity, which could be analogous, in insects. This provides further support to the functional analogies of vertebrate and invertebrate endocrine systems postulated by different authors, and underlines once again the suitability of insects as models for neuroendocrine research (see Scharrer, 1987). Acknow[edRements-Thanks are due to Dr Francisco Camps for critical reading of the manuscript. Funds from CAICYT (project 84-0087) and CSIC (project 263-85) are also gratefully acknowledged.
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