Autocrine activation of ecdysteroidogenesis in the prothoracic glands of the silkworm, Bombyx mori

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Journal of Insect Physiology 53 (2007) 538–549 www.elsevier.com/locate/jinsphys

Autocrine activation of ecdysteroidogenesis in the prothoracic glands of the silkworm, Bombyx mori Shi-Hong Gu Department of Zoology, National Museum of Natural Science, 1 Kuan Chien Road, Taichung, Taiwan 404, ROC Received 30 June 2006; received in revised form 14 February 2007; accepted 14 February 2007

Abstract Ecdysteroidogenesis in the prothoracic glands is activated by the neuropeptide, prothoracicotropic hormone (PTTH). The present study demonstrates autocrine activation of ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori. Using both a longterm in vitro organ culture system and an ecdysteroid radioimmunoassay, it was found that either decreasing the incubation volume, from 100 to 5 ml, or increasing the number of glands incubated per drop (50 ml) from 1 to 5 significantly increased ecdysteroid secretion. Prothoracic gland-conditioned medium was used to clarify the autocrine factor. The results showed that activation of ecdysteroidogenesis by the prothoracic gland-conditioned medium appeared to be dose dependent and a dramatic increase in ecdysteroid secretion was observed after 6 h of incubation in the conditioned medium. Moreover, it appeared that autocrine activation occurred when glands were incubated in large volumes of incubation medium and during a short incubation period, indicating that the factor may exert its action in situ at some specific developmental stages. This tropic factor was further characterized, and it was found that the factor seemed to be heat-stable, with a molecular weight estimated to be between 1000 and 3000 Da. Injection of the concentrated putative autocrine factor into day 5 last instar larvae greatly increased ecdysteroidogenic activity of the prothoracic glands compared to those injected with saline, indicating the possible in vivo function of the present factor. r 2007 Elsevier Ltd. All rights reserved. Keywords: Bombyx mori; Autocrine ecdysiotropic factor; Ecdysteroid secretion; Long-term incubation; Organ culture; Prothoracic gland; In vitro; In vivo function

1. Introduction Ecdysteroids, synthesized by insect prothoracic glands, play important roles in directing insect growth and development, with the major ecdysteroid peaks eliciting molting and metamorphosis (Gilbert et al., 2002). It is generally accepted that ecdysteroidogenesis by the prothoracic glands is stimulated by the neuropeptide, prothoracicotropic hormone (PTTH). Since Kopec (1922) first demonstrated that the larval brain of the gypsy moth, Lymantria dispar, secretes a factor needed for molting and metamorphosis, attempts have been made to purify and characterize PTTH and its mode of action (Gilbert et al., 2002).

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In Bombyx mori, ecdysteroidogenesis of the prothoracic glands undergoes specific developmental changes during the penultimate and last larval instars (Okuda et al., 1985; Gu and Chow, 2005b; Gu et al., 1996, 1997, 2000). The prothoracic glands of the early 4th larval instar produce detectable ecdysteroid levels and are stimulated by PTTH. However, glands of the 1st stages of the last larval instar cannot produce detectable ecdysteroid levels and show no response to PTTH (Okuda et al., 1985; Gu et al., 1996, 1997, 2000; Takaki and Sakurai, 2003; Sakurai, 2005). We previously demonstrated that PTTH signal transduction pathways undergo specific developmental changes, with a deficiency in transduction in prothoracic gland cells occurring during the early last instar (Gu et al., 1996). During the 1st stage of the last larval instar, PTTH is released from the brain–corpus cardiacum–corpus allatum (BR–CC–CA) complex (Shirai et al., 1993; Dai et al., 1995; Gu et al., 1996), but the prothoracic glands show no

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increase in either cAMP levels or steroidogenesis in response to stimulation by PTTH (Gu et al., 1996). Thus, it is clear that both PTTH release and successful PTTH signal transduction are crucial for ecdysteroidogenesis. Recent studies from our laboratory have shown that such stage-specific changes in ecdysteroid production play critical roles in regulating larval molting and metamorphosis, with the inactivation of prothoracic glands being a cue to commence the sequential developmental events leading to very low ecdysteroid levels, inactivation of the corpora allata, and larval–pupal transformation (Gu and Chow, 1996, 2005b; Gu et al., 1996). Moreover, recently we have focused on the regulation of cell growth of both the prothoracic glands and corpora allata (Gu and Chow, 2001, 2003, 2005a). We reported that DNA synthesis in prothoracic gland cells of B. mori undergoes specific developmental changes during the 3rd, 4th, and last larval instars: the dramatic increases in DNA synthesis during the early (for the 3rd and 4th instars) or middle (for the last instar) stages precede the major increase in ecdysteroidogenesis during the later stages (Gu and Chow, 2001, 2005a). We also demonstrated that a novel autocrine factor activates DNA synthesis of gland cells in vitro (Gu, 2006). Our results showed that DNA synthesis of prothoracic gland cells incubated in a small volume of medium (10 ml) dramatically increased compared with those incubated in a large volume (50 ml) (Gu, 2006), implying the possibility that ecdysteroid secretion by the silkworm prothoracic glands may differ when the glands are incubated in different incubation volumes. The present study indeed confirmed this view. Herein, I demonstrate the autocrine activation of ecdysteroidogenesis by silkworm prothoracic glands both in vitro and in vivo. 2. Materials and methods 2.1. Experimental animals Silkworm larvae were reared on fresh mulberry leaves at 25 1C under a 12-L:12-D photoperiod. Animals were staged on the day of ecdysis to the last instar, and this day was designated day 0 of the last instar. Larvae began wandering on day 7 of the last larval instar, and pupation occurred on day 11. 2.2. Collection of prothoracic gland- and other tissuepreconditioned media Prothoracic glands from day 2 last instar larvae were dissected out under lepidopteran saline (Gu et al., 1995a, b), and then rinsed with the same saline 3 times to avoid contamination by hemolymph. The glands were then incubated for 2 days in 10 ml of Grace’s medium. After incubation, the glands were discarded, and the medium was sterilized by filtration (MFS-13, pore size 0.2 mm, Advantec MFS, CA, USA) and stored at 20 1C until required. Control medium was similarly incubated and prepared, but

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without addition of the prothoracic glands. All incubations were carried out in 96-well culture plates sealed with a parafilm membrane and maintained in a humidified incubator at 25 1C. Glands from day 2 last instar larvae were selected to obtain conditioned medium, because ecdysteroid levels released during the 2-day incubation period are low, but there is high autocrine activity (Gu, 2006, see Section 3). To study whether or not other tissues also release a stimulatory factor in vitro to activate ecdysteroidogenesis of prothoracic glands, wing discs, a small piece of silk glands and fat body from day 2 last instar larvae were preincubated in a small volume similar to the prothoracic glands (10 ml). After 48 h of incubation, each tissue was then removed and the medium was collected, sterilized by filtration, and used to incubate prothoracic glands from day 7 last instar larvae in a large volume (50 ml/gland). 2.3. Reagents 20-Hydroxyecdysone and dibutyryl cAMP (dbcAMP) were from Sigma (St. Louis, MO, USA). Grace’s insect cell culture medium was purchased from Gibco (Invitrogen, Carlsbad, CA, USA). [23,24-3H] Ecdysone was purchased from New England Nuclear (Boston, MA, USA). Recombinant B. mori PTTH (O’Reily et al., 1995; Gu et al., 1998; Chen and Gu, 2006) was diluted 200 times with medium for use in this study. 2.4. In vitro incubation of prothoracic glands and radioimmunoassay (RIA) of ecdysteroids Prothoracic glands from day 7 last instar larvae were dissected out under lepidopteran saline (Gu et al., 1995b). After dissection, the glands were preincubated in 100 ml of medium for 1 h, and then transferred to control or prothoracic gland-conditioned media. Most of the incubations in the present study were maintained for 24 h except when specifically stated otherwise. After incubation, the released ecdysteroids in the medium were determined by an RIA according to procedures described in previous studies (Takeda et al., 1986; Gu et al., 1995a; Gu and Chow, 1996). The assay was calibrated using 20-hydroxyecdysone as the standard. The antiserum has an approximate binding ratio of 2.5:1 for 20-hydroxyecdysone to ecdysone (Takeda et al., 1986). The detection limit of the RIA was 0.03 ng. For determining hemolymph ecdysteroids, hemolymph was collected at each defined time and stored at 20 1C before use. Ecdysteroids were extracted from hemolymph with methanol; after centrifugation, the supernatants were evaporated to dryness. 2.5. Preparation of the extracts of prothoracic glands To obtain extracts of prothoracic glands, freshly dissected prothoracic glands of day 2 last instar larvae and glands that had been incubated in 10 ml of medium for

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48 h were separately homogenized in saline, and the homogenate was immediately heated in a water bath to 100 1C for 2 min to inactivate the peptide-degrading enzymes. After centrifugation (10,000g, for 10 min), the supernatant was sterilized by filtration (through a filter with a pore size of 0.2 mm). Prothoracic gland extract was then dissolved in the medium for incubation. 2.6. Heating and protease digestion To characterize the nature of the prothoracic glandpreconditioned medium, the medium was heated in a water bath to 100 1C for 2 min or incubated with trypsin (2 mg/ml, type II-S, Sigma Chemical) at 37 1C for 2 h. After the trypsin treatment, a soybean trypsin inhibitor was added to the medium to stop the action of trypsin. The soybean trypsin inhibitor-inactivated trypsin was also used as the control. To characterize the nature of the wing disc-preconditioned medium, the medium was incubated with proteinase K (at a final concentration of 1 mg/ml, Sigma Chemical) at 37 1C for 4 h. After proteinase K treatment, the mixture was boiled for 4 min. Heatinactivated proteinase K was also used as the control. 2.7. Molecular mass estimation Prothoracic gland-preconditioned medium (1 ml) was placed in the upper chamber of a microconcentrator (Microcon Model YM-3, Amicon, Danvers, MA) and centrifuged at room temperature for 100 min at 12,000g. According to the manufacturer’s literature, the fraction remaining in the upper chamber after centrifugation should have an MrXof 3000 Da, while the molecules entering the lower chamber (filtrate) should have an Mr of p3000 Da. The resulting fractions in each of the chambers were assayed for ecdysiotropic activity. To examine whether or not the Mr of the putative ecdysiotropic factor exceeded 1000 Da, 800 ml of prothoracic glandconditioned medium was extensively dialyzed using a CelluSep H1 membrane with a nominal MWCO of 1000 Da (Membrane Filtration Products, TX, USA) in distilled water at 4 1C overnight. The fraction remaining in the bags was evaporated to dryness under reduced pressure and then dissolved in medium to test for ecdysiotropic activity. 2.8. Injection of concentrated autocrine factor To determine whether or not the putative autocrine factor has biological function in vivo, day 5 last instar larvae were injected with different doses of concentrated autocrine factor dissolved in 10 ml saline or with 10 ml saline only as the control. The doses of 1, 2, and 5 prothoracic gland equivalents were used for each injection time. One prothoracic gland equivalent means that it initially contained 10 ml of the prothoracic gland-conditioned medium. The same injection was repeated 1 time after a

3 h interval. Three hours after the 2nd injection, the prothoracic glands were dissected out and then incubated in medium for 2 h. After incubation, ecdysteroid levels in the medium were determined by the RIA. To prepare the concentrated autocrine factor from prothoracic gland-conditioned medium, 1 ml of conditioned medium was extensively dialyzed using a CelluSep H1 membrane in distilled water at 4 1C overnight. The fraction remaining in the bag was evaporated to dryness under reduced pressure and then dissolved in 150 ml of saline for injection. The concentrated autocrine factor was then diluted with saline according to different injection doses (1, 2, and 5 prothoracic gland equivalents).

3. Results 3.1. Effects of the incubation volume of the medium on ecdysteroid secretion by the prothoracic gland Our recent study showed that an increase in prothoracic gland DNA synthesis during silkworm development was correlated with an increase in ecdysteroidogenic activity (Gu and Chow, 2005a). Moreover, it was demonstrated that silkworm prothoracic gland DNA synthesis appeared to be stimulated by an autocrine factor and that either decreasing the incubation volume, from 100 to 5 ml, or increasing the number of glands incubated per drop (50 ml), from 1 to 6, significantly increased gland cell DNA synthesis (Gu, 2006). Those results prompted me to examine whether or not ecdysteroid secretion changed with different incubation media. Thus, initial experiments were conducted to examine whether or not ecdysteroid secretion by the prothoracic glands was affected by different incubation volumes. Fig. 1A shows the effects of different volumes of incubation medium (100, 50, 20, 10, and 5 ml) on ecdysteroid secretion during the 1st 24 h incubation period. Clearly, gradually decreasing the incubation volume greatly increased ecdysteroid secretion (Fig. 1A). Because a long-term cultivation system was used in the present study, it is possible that when the glands were incubated in very small volumes of medium for 24 h, ecdysteroid metabolism may have greatly changed according to the incubation volume, thus leading to different levels of immunoreactive ecdysteroids. To rule out this possibility, after incubation in different volumes of medium for the 1st 24 h, each gland was rinsed 3 times with saline and then transferred to the same 50 ml volume of medium. The incubations were continued for an additional 2 h. Fig. 1B shows that the increased ecdysteroid secretion was maintained through the 2nd 2 h incubation period in glands which had been incubated in the small volume of medium for the 1st 24 h. This clearly shows that ecdysteroidogenic activity was indeed enhanced by incubation in the small volume of medium for the 1st 24 h.

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Fig. 1. Effects of different incubation volumes on ecdysteroid secretion by prothoracic glands from day 7 last instar larvae. Glands were incubated in 5, 10, 20, 50, or 100 ml Graces medum. After 24 h of incubation, each gland was individually transferred to an identical 50 ml volume of medium, and the incubation continued for a 2nd 2 h period. (A) Ecdysteroids released into the medium during the 1st 24 h period. (B) Ecdysteroids released into the medium during the 2nd 2 h period. a–e indicate ecdysteroid levels released by the glands during the 2nd 2 h, which had been incubated for the 1st 24 h in 100, 50, 20, 10, and 5 ml medium, respectively. Each value is the average7SEM (n ¼ 5). *Indicates a significant difference compared with 100 ml of medium (for A) or with a (for B) (by Student’s t-test, po0.01).

3.2. Effects of the numbers of glands incubated in the same volume of medium The above results show that ecdysteroid secretion by prothoracic glands incubated in small volumes (5 or 10 ml) was significantly higher compared with that of glands incubated in large volumes (50 or 100 ml). The effects of increasing the number of glands incubated in the same volume of medium were further examined. As shown in Fig. 2A, glands cultured in groups (5 glands in a 50 ml drop) resulted in significantly higher levels of ecdysteroid secretion than those cultured individually in 50 ml drops (5.65 and 0.58 ng/gland for 5 glands together and 1 gland/ 50 ml medium, respectively). To rule out the possibility that when the glands were incubated in a crowded condition for 24 h, higher ecdysteroid secretion may have been due to changed ecdysteroid metabolism, after incubation with different numbers of glands for the 1st 24 h period, each gland was rinsed 3 times with saline and then transferred to

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Fig. 2. Effects of group incubation of glands on ecdysteroid secretion by prothoracic glands. Glands were incubated with different numbers of glands (1, 2, and 5) for the 1st 24 h period, and each gland was then individually transferred to 50 ml of medium, and the incubations were maintained for an additional 2 h period. (A) Ecdysteroids released into the medium during the 1st 24 h period. (B) Ecdysteroids released into the medium during the 2nd 2 h period. a–c indicate ecdysteroid levels released by the glands during the 2nd 2 h period, which had been incubated for the 1st 24 h period individually or with 2 or 5 glands together, respectively. For the group incubation, ecdysteroid levels were calculated as ng/gland. Each value is the average7SEM (n ¼ 5). *Indicates a significant difference compared to levels individually incubated (for A) or to a (for B) (by Student’s t-test, po0.01).

the same 50 ml volume of medium, and the incubations were maintained for an additional 2 h period. Similar increased levels of ecdysteroid secretion as for glands incubated in different volumes of medium (Fig. 1) were also observed for the 2nd 2 h incubation period with glands, which had been incubated in a 50 ml drop with 5 glands together for the 1st 24 h period (Fig. 2B).

3.3. Effects of ecdysone and 20-hydroxyecdysone To rule out the possibility that ecdysteroid levels in the incubation medium may have affected ecdysteroid secretion during the subsequent incubation period, prothoracic glands from day 7 last instar larvae were incubated in media containing different concentrations (20 and 200 ng/ml) of either ecdysone or 20-hydroxyecdysone. After incubation for the 1st 24 h, each gland was rinsed 3

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times with saline and then transferred to the same 50 ml volume of control medium, and the incubations were maintained for an additional 2 h. As shown in Fig. 3, inclusion of ecdysone or 20-hydroxyecdysone in the incubation medium during the 1st 24 h did not show a stimulatory effect on ecdysteroidogenesis during the 2nd 2 h incubation period. 3.4. Continued ecdysteroid secretion of prothoracic glands during long-term incubation periods and their responsiveness to PTTH and dbcAMP To verify the present long-term cultivation system, day 7 prothoracic glands were incubated in 10 or 50 ml of medium. Incubation was continued for more than 4 days. The basal ecdysteroid secretion level and changes in responsiveness to either PTTH or dbcAMP were carefully examined. As shown in Fig. 4A, during the 1st 2 h incubation period, no difference in ecdysteroid secretion was observed between the 10- and 50 ml volumes of incubation medium. However, a difference in ecdysteroid secretion was observed when the incubation was continued for the 2nd 24 h period: much more ecdysteroid was secreted by the prothoracic glands incubated in the 10 ml volume of medium compared to those incubated in the 50 ml volume of medium (4.18 and 0.40 ng/gland for 10 and 50 ml of medium, respectively). Higher ecdysteroid secretion in glands incubated in 10 ml of medium continued through the 3rd 24 h period. However, during the 4th and 5th 24 h periods, no differences in ecdysteroid secretion were observed between the 10- and 50 ml incubation volumes, although ecdysteroid secretion was still detectable for both incubation volumes. To examine whether or not glands incubated in my long term cultivation system are healthy, after incubation in different volumes of medium

Fig. 3. Effects of ecdysone (or 20-hydroxyecdysone) on ecdysteroid secretion during the 2nd 2 h incubation period. Prothoracic glands were incubated in 50 (A) or 10 ml (B) of control medium or 50 ml of medium containing 20 ng/ml 20-hydroxyecdysone (C), 200 ng/ml 20-hydroxyecdysone (D), 20 ng/ml ecdysone (E), and 200 ng/ml ecdysone (F) for the 1st 24 h period, and each gland was rinsed and then transferred to 50 ml of the control medium, and the incubations were maintained for a 2nd 2 h incubation period. Each value is the average7SEM (n ¼ 5). *Indicates a significant difference compared with A (by Student’s t-test, po0.01).

Fig. 4. Effects of different incubation volumes on ecdysteroid secretion by the prothoracic glands. (A) Ecdysteroid secretion during a long-term incubation. Prothoracic glands were incubated in 10 (10) or 50 (50) ml of medium, respectively. After each defined incubation period (the 1st for 2 h, the 2nd for 24 h, the 3rd for 24 h, and the 4th for 24 h), each gland was transferred to fresh medium (the volume of the incubation medium was kept the same as in the previous incubation). (B, C) Effects of PTTH and dbcAMP on ecdysteroid secretion by prothoracic glands that had been incubated in 10 (B) or 50 ml (C) of medium for different lengths of time. After the defined incubation periods, each gland was then individually transferred to 50 ml of medium containing either PTTH (PTTH), 10 mM dbcAMP (dbcAMP), or control medium (control), and the incubations were continued for an additional 4 h period. Each value is the average7SEM (n ¼ 5).

for the 1st day, each gland was rinsed 3 times with saline and then transferred to the same 50 ml volume of medium, and the responsiveness to stimulation by either PTTH or dbcAMP was further investigated. Fig. 4B (the 2nd 4 h period) shows that the glands which had been incubated in 10 ml of medium for the 1st 24 h showed responsiveness to either PTTH or dbcAMP during the 2nd 4 h incubation. This responsiveness was also observed in glands that had been incubated in 50 ml of medium for the 1st 24 h (Fig. 4C, the 2nd 4 h), although the basal ecdysteroid secretory activity was low. This responsiveness was maintained after incubation for 2 (Fig. 4B and C, the 3rd 4 h) or 3 days

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(Fig. 4B and C, the 4th 4 h). These results clearly show that in the present long-term cultivation system, not only did basal ecdysteroid secretion continue, but also the cAMPdependent steroidogenic apparatus remained functional. 3.5. Effects of prothoracic gland-conditioned medium on ecdysteroid secretion Prothoracic gland-conditioned medium was used to test whether or not prothoracic glands secrete the autocrine factor which stimulates their own ecdysteroid secretion. Prothoracic glands from day 2 last instar larvae were preincubated in a small volume (10 ml). After a 48 h incubation, the glands were removed and the medium was collected, sterilized by filtration, and then used to incubate additional prothoracic glands from day 7 last instar larvae in a large volume (50 ml/gland). The results (Fig. 5A) showed that as with glands incubated in a small volume of medium (10 ml), levels of ecdysteroid secretion by

Fig. 5. Dose- and time-dependent effects of conditioned medium on ecdysteroidogenesis by prothoracic glands. (A) Dose-dependent effects. Medium preconditioned by a 48 h incubation of prothoracic glands from day 2 last instar larvae was collected and diluted with control medium to establish a dose–response curve. (B) Time-dependent effects. Each gland was incubated in 100% conditioned medium or in control medium, and after being incubated for a defined period, ecdysteroids released into the medium were determined. Each value is the average7SEM (n ¼ 5). *Indicates a significant difference compared with those incubated in control medium (by Student’s t-test, po0.01).

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glands incubated in this conditioned medium were much higher compared with those in control medium although the medium volume was the same (50 ml) (14.42 and 0.48 ng/gland for the conditioned and control media, respectively). The initial ecdysteroid level in the 50 ml of conditioned medium was only 0.8 ng. The net increase after subtracting the initial (before the 2nd incubation) ecdysteroid level was 14.42 ng/gland. Using the above prothoracic gland-conditioned medium, the dose- and time-dependent effects on ecdysteroid secretion were examined. Medium preconditioned with a 48 h incubation of prothoracic glands was collected and diluted to establish a dose–response curve. As shown in Fig. 5A, the stimulation of ecdysteroid secretion by prothoracic glands was dependent on the concentration of the conditioned medium. The addition of 50% conditioned medium still showed stimulatory effects. The time course of stimulation indicated that a high level of stimulation existed after 6 h of incubation (Fig. 5B). In other experiments, glands from day 2 last instar larvae were preincubated in medium for an initial 5 h period and then transferred to another 10 ml volume of medium, and the incubation was continued for an additional 19 h. After 19 h of incubation, glands were transferred to another 10 ml of medium, and the incubation was continued for another 24 h period. The medium was then collected and tested for the stimulatory effects of ecdysteroid secretion. As shown in Fig. 6, the preincubated media showed strong stimulatory effects on ecdysteroid secretion, indicating that the glands may have continuously been releasing the autocrine factor. The effects of freshly dissected prothoracic glands

Fig. 6. Effects of different preincubation times and prothoracic gland extract on ecdysteroidogenesis by prothoracic glands. Glands incubated in control medium (control) or in prothoracic gland-conditioned medium for which glands from day 2 last instar larvae had been preincubated for the 1st 5 h (1st 5 h) or for the 2nd 19 h (2nd 19 h) or for the 3rd 24 h (3rd 24 h); Extract A and B, glands incubated in medium that contained 1 equivalent of extract from freshly dissected glands (extract A) or from glands that had been incubated for 48 h in 10 ml of medium (extract B). After incubation for 24 h, ecdysteroids released into the medium were determined. Each value is the average7SEM (n ¼ 5). Ecdysteroid values for conditioned medium or for incubation with gland extract are the net ecdysteroid value after subtracting the initial (before the 2nd incubation or contained in the extract) ecdysteroid levels. *Indicates a significant difference compared with the controls (by Student’s t-test, po0.01).

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and glands that had been incubated in 10 ml of medium for 48 h were also examined. Results showed that neither set of glands had any stimulatory effects on ecdysteroidogenesis. These results indicate that the majority of the autocrine factor may have been secreted as soon as it was synthesized, i.e., that it was not retained in the glands. Ecdysteroidogenic activities of prothoracic glands of insects have often been studied using short-term incubation periods (of 1 or 2 h) (Gilbert et al., 2002). Subsequent experiments were used to determine whether or not the autocrine factor was released during the short-term incubation period. Glands from day 2 last instar larvae were preincubated in 10 ml of medium for the 1st 2 h period. Each gland was rinsed 3 times with saline and then transferred to another 10 ml of medium, and the incubation was maintained for an additional 2 h period. After the 2nd 2 h incubation period, the medium was collected and tested for the stimulatory effects of ecdysteroid secretion. As shown in Fig. 7, these preincubated media showed strong stimulatory effects on ecdysteroid secretion, indicating that the glands may have released the autocrine factor even during the short incubation period. In addition, the above results showed that when glands were incubated in large volumes (100 or 50 ml medium), ecdysteroidogenic activity was lower compared with those incubated in small volumes (10 or 5 ml medium, Fig. 1).

Fig. 7. Effects of different preincubation times and different preincubation volumes on ecdysteroidogenesis by prothoracic glands. Glands incubated in control medium (C) or in prothoracic gland-conditioned medium for which glands from day 2 last instar larvae had been preincubated for the 1st 2 h (1st 2 h) or for the 2nd 2 h (2nd 2 h). 50 ml CM and 100 ml CM, glands incubated in prothoracic gland-conditioned medium for which glands from day 2 last instar larvae had, respectively, been preincubated in 50 or 100 ml of medium for 48 h. After incubation for 24 h, ecdysteroids released into the medium were determined. Each value is the average7SEM (n ¼ 5). Ecdysteroid values for conditioned medium are the net ecdysteroid value after subtracting the initial (before the 2nd incubation) ecdysteroid levels. *Indicates a significant difference compared with the controls (by Student’s t-test, po0.05).

This result does not rule out the possibility that when glands are incubated in a large volume of medium, no autocrine factor is released. To clarify whether or not the autocrine factor is released even when glands are incubated in a large volume of medium, further experiments were conducted. Glands from day 2 last instar larvae were, respectively, preincubated in 100 or 50 ml medium for 48 h. After incubation, each conditioned medium was collected, and tested for the stimulatory effects of ecdysteroid secretion. As shown in Fig. 7, the preincubated media in which glands from day 2 last instar larvae had been incubated in 50 or 100 ml of medium for 48 h showed stimulatory effects on ecdysteroid secretion. This result indicates that when glands are incubated in large volumes (50 or 100 ml), they still release the autocrine factor. 3.6. Partial characterization of the autocrine ecdysiotropic factor The nature of this autocrine factor was further investigated. The results (Fig. 8) showed that this factor appears to be heat-stable. After treatment at 100 1C for 2 min, the factor remained active. Moreover, after incubation with trypsin at 37 1C for 2 h, the stimulatory activity of this factor remained active. Proteinase K treatment and different pH conditions of the medium did not affect the activity of the factor (Gu, unpublished data). The molecular weight of the autocrine factor was further estimated. Microconcentrator (Microcon Model YM-3) filtration allowed most of the detectable activity of the factor to pass into the filtrate; slightly higher activity

Fig. 8. Partial characterization of a putative autocrine factor. Control, glands incubated in control medium; conditioned medium, glands incubated in prothoracic gland-conditioned medium without further treatment; trypsin, glands incubated in conditioned medium that had previously been treated with trypsin; heat, glands incubated in conditioned medium that had previously been heated in a water bath to 100 1C for 2 min; filtrate, glands incubated in conditioned medium that had passed through Microcon YM-3 filters; Retentate, glands incubated in the conditioned medium that had not passed through the Microcon YM-3 filters. Each value is the average7SEM (n ¼ 5). Ecdysteroid value for conditioned medium was the net ecdysteroid value after subtracting the initial (before the 2nd incubation) ecdysteroid levels. *Indicates a significant difference compared with those incubated in the control medium (by Student’s t-test, po0.01).

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compared to that of the control was also detected in the retentate (Fig. 8). Data from the Amicon microconcentrators indicated that the Mr of the autocrine factor is likelyo3000 Da. To examine whether or not the Mr of the putative autocrine factor exceeds 1000 Da, 800 ml of prothoracic gland-conditioned medium was placed in CelluSep H1 regenerated cellulose tubular membrane dialysis bags, and the medium was extensively dialyzed in distilled water at 4 1C overnight, evaporated to dryness under reduced pressure, and then dissolved in medium to determine its ecdysiotropic activity. The results showed that the fraction maintained in the membrane dialysis bags still showed ecdysiotropic activity (0.6970.11 and 1.2070.15 ng/gland for the control and putative autocrine factor-treated glands, respectively; n ¼ 5;7SEM), indicating that the Mr of the factor may be 41000 Da. In addition, the stimulatory effects of the fraction maintained in the membrane dialysis bag were lower compared to that of untreated conditioned medium, implying that the factor with an MW of o1000 Da might also be involved in ecdysiotropic activity. The true Mr cannot be ascertained without further studies. 3.7. In vivo function of the autocrine ecdysiotropic factor To study whether or not the putative autocrine factor can exert biological functions in vivo, the autocrine factor from prothoracic gland-conditioned medium was concentrated using a CelluSep H1 membrane as described in ‘‘Methods’’. Day 5 last instar larvae were injected with concentrated factor dissolved in 10 ml saline or with only 10 ml saline as the control. After 1 repeated injection after a 3 h interval, the prothoracic glands were dissected out and their ecdysteroidogenic activities were determined. Results (Fig. 9) showed that during a 2 h incubation, ecdysteroidogenic activity from the prothoracic glands of larvae injected with the concentrated autocrine factor dosedependently increased compared to those injected with 10 ml saline only. Net increases in ecdysteroid secretion of 62%, 48%, and 28% compared with the control were observed for injections of 5, 2, and 1 prothoracic gland equivalents, respectively. Determination of hemolymph ecdysteroid levels 6 h after the 1st injection showed that higher ecdysteroid levels were observed in larvae injected with concentrated autocrine factor compared to those injected with saline only (23.5, 20.3, 17.1, and 13.1 ng/ml for injections of 5, 2, and 1 prothoracic gland equivalents, and saline injection only, respectively; n ¼ 5). These results conclusively show that the putative autocrine factor secreted by prothoracic gland cells in vitro can exert a biological function in vivo. 3.8. Effects of other tissue-conditioned media on ecdysteroid secretion Finally, to examine whether or not other tissues also release a stimulatory factor in vitro which activates

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Fig. 9. Dose-dependent effects of injection of concentrated autocrine factor on the in vitro ecdysteroid secretion by prothoracic glands. Day 5 last instar larvae were injected with different doses of concentrated autocrine factor. After 1 repeated injection at a 3 h interval, prothoracic glands were dissected out, and their ecdysteroidogenic activities were determined. Control, each larva was injected with saline only; injection of autocrine factor, each larva was injected with concentrated autocrine factor from conditioned medium; 1, 2, and 5, each larva was injected with saline, respectively, containing 1, 2, and 5 prothoracic gland equivalents for each injection time. After incubation, ecdysteroids released into the medium were determined. Each value is the average7SEM (n ¼ 5). *Indicates a significant difference compared with the controls (by Student’s t-test, *po0.05; **po0.01).

ecdysteroidogenesis by the prothoracic glands, wing discs, a small piece of silk glands and fat body from day 2 last instar larvae were preincubated in a small volume (10 ml) of medium. After a 48 h incubation, each tissue was removed, and the medium was collected, sterilized by filtration, and used to incubate prothoracic glands from day 7 last instar larvae in a large volume (50 ml per gland). The results (Fig. 10) showed that when the prothoracic glands were incubated in wing disc- or fat body-conditioned media, greatly increased ecdysteroid secretion was observed compared with those in control medium. In contrast, silk gland-conditioned medium showed no stimulatory effects. Moreover, when wing disc-conditioned medium was treated with proteinase K, the stimulatory effect was greatly decreased. The molecular weight of the stimulatory factor from wing disc-conditioned medium was further estimated. Higher activity was detected in the retentate compared to that in the filtrate (Fig. 10). Data from the Amicon microconcentrators indicated that the Mr of the factor from wing disc-conditioned medium might exceed 3000 Da. The possibility thus exists that there is an additional paracrine activator of ecdysteroidogenesis and the factor from wing-disc-conditioned medium may differ from that from the prothoracic glands. 4. Discussion The present study clearly shows that the prothoracic glands from silkworm larvae synthesize and secrete an autocrine factor that can activate the ecdysteroid secretion

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Fig. 10. Effects of other tissue-conditioned medium on ecdysteroid secretion and its partial characterization. Control, glands incubated in control medium; WD, glands incubated in wing disc-conditioned medium; FB, glands incubated in fat body-conditioned medium; SG, glands incubated in silk gland-conditioned medium; Retentate, glands incubated in the wing disc-conditioned medium that had not passed through a Microcon YM-3 filter; filtrate, glands incubated in wing disc-conditioned medium that had passed through a Microcon YM-3 filter; Proteinase K, glands incubated in wing disc-conditioned medium that had previously been treated with proteinase K; Inactive proteinase K, glands incubated in wing disc-conditioned medium that had previously been treated with inactivated proteinase K. Each value is the average7SEM (n ¼ 5). *Indicates a significant difference compared with those incubated in control medium (by Student’s t-test, po0.01).

of glands both in vivo and in vitro. Although it has been demonstrated in experiments with Locusta migratoria (Charlet and Hoffman, 1982), Manduca sexta (Keightley et al., 1990; Kulesza et al., 1994; Rybczynski and Gilbert, 1994), and Periplaneta americana (Richter and Baumann, 1997) that prothoracic glands secrete some proteins besides ecdysteroids, to my knowledge, this is the 1st report to demonstrate that prothoracic glands secrete an autocrine ecdysiotropic factor in vitro. The results showed that incubation of glands in prothoracic gland-conditioned medium or culturing groups of glands leads to a substantial dose-dependent increase in ecdysteroid secretion during a long-term incubation period (24 h). Moreover, the present study clearly shows that the putative autocrine factor may have an in vivo biological function: it greatly increased ecdysteroidogenic activity of prothoracic glands when larvae were injected with concentrated putative growth factor. More than 50 years ago, Williams first demonstrated that prothoracic glands could be triggered by the prothoracic gland hormone itself (Williams, 1952). The present study not only re-confirms the above result, but clearly indicates that a novel factor other than ecdysteroids may be involved in activation of the prothoracic glands by the glands themselves. Feedback regulation of prothoracic glands by ecdysteroids has recently been well documented (Sakurai, 2005). Ecdysteroids not only play an important role in regulating insect growth and development, but also have feedback action on ecdysteroidogenesis in both prothoracic glands

and testes (Beydon and Lafont, 1983; Loeb et al., 1986; Sakurai and Williams, 1989; Jiang and Koolman, 1999; Gilbert et al., 2002). In M. sexta, prothoracic glands can either be stimulated or inhibited by ecdysteroids, depending on both the secretory activity of the glands and the effective levels of ecdysteroids (Sakurai and Williams, 1989). Rhythmic autocrine activity was also reported in ecdysteroid production in a cultured insect IAL-PID2 cell line (Mesnier et al., 2000). Recently, using a long-term in vitro cultivation system, Mizoguchi and Kataoka (2005) demonstrated that inactive prothoracic glands from day 0 last instar silkworm larvae could be spontaneously activated without stimulation by PTTH. In the present study, it was found that either decreasing the incubation volume, from 100 to 5 ml, or increasing the number of glands incubated per drop (50 ml), from 1 to 5, significantly increased ecdysteroid secretion. The prothoracic glandconditioned medium also showed stimulatory effects on ecdysteroidogenesis in a dose-dependent manner. It is assumed that a novel factor other than ecdysteroids is involved in autocrine activation of ecdysteroidogenesis by silkworm prothoracic glands due to the following reasons: (1) During the 1st 3 days of the last larval instar in silkworms, the prothoracic glands, which are inactive in ecdysteroidogenesis (Okuda et al., 1985; Gu et al., 1996, 1997), also synthesized and secreted the autocrine factor that stimulated ecdysteroidogenesis by glands from the later stages of the last instar larvae (Fig. 5, Gu, unpublished data). (2) The present results showed that the Mr of the autocrine ecdysiotropic factor might be 41000 Da, which differs from ecdysteroids. Determination of ecdysteroid levels showed that no immunoreactive ecdysteroids existed in the CelluSep H1 dialysis bag after dialysis. (3) Injection of concentrated autocrine factor into day 5 last instar larvae led to increases in ecdysteroidogenesis of the prothoracic gland, and hemolymph ecdysteroid levels were also greatly increased 6 h after the 1st injection, indicating that neither ecdysteroids, nor another artifact, but a true ecdysiotropic factor is involved in the activation of ecdysteroidogenesis. (4) As shown in Figs. 1B and 2B, the greatly increased ecdysteroid secretion by prothoracic glands was maintained for the 2nd 2 h incubation period after removal of exposure to either the small volume of medium (Fig. 1B) or a crowded incubation condition (Fig. 2B). Inclusion of ecdysone or 20-hydroxyecdysone into the incubation medium during the 1st 24 h incubation period showed no such a stimulatory effect on ecdysteroidogenesis during the subsequent incubation period (Fig. 3). In addition, as shown in Fig. 4, the glands which had been incubated in 10 ml of medium for the 1st 24 h incubation period showed activation responsiveness to either PTTH or dbcAMP during the 2nd 4 h incubation period. Activation responsiveness was also observed in glands that had been incubated in 50 ml of medium for the 1st 24 h incubation period, although their basal ecdysteroid secretory activity was low. This activation responsiveness was maintained after incubation for 2 or 3 days. This result

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indicates that not only a basal but also a PTTH-cAMPresponsive steroidogenic apparatus were maintained in the present cultivation system. From these results, it is assumed that during the present long-term cultivation condition, the glands remained healthy. However, in the present study, prothoracic glands were incubated in a very small volume of medium or in a crowded condition for more than a day, and the possibility exists that accumulation of some intermediate metabolites or other artifacts may have occurred, and this may have led to different levels of immunoreactive ecdysteroids. Further study is needed to carefully check for the possible occurrence of some intermediate metabolites or some artifacts and to examine whether or not this is related to differences in ecdysteroid secretion. By contrast, the results that either decreasing the incubation volume, from 100 to 5 ml, or increasing the number of glands incubated per drop (50 ml), from 1 to 5, significantly increased ecdysteroid secretion do not rule out the possibility that when glands were incubated in a large volume, no autocrine factor exerts its action. This view is supported by the present study. Glands showed an activation response to preincubation medium that had first been incubated in 100 or 50 ml medium, compared with that incubated in control medium (Fig. 7). Moreover, the present study showed that glands may release the autocrine factor even during a short incubation period (2 h, Fig. 7). These results suggest that in a large volume of incubation medium and even during a short incubation period, a low but sustained autocrine factor may be released and exerts its action and that high stimulatory effects of preconditioned medium that had first been incubated in 10 ml medium may just be due to the presence of a concentrated autocrine factor in the medium. Thus, considering the fact that in the in vivo physiological condition, prothoracic glands are bathed in a large amount of hemolymph, it is supposed that an autocrine factor may also play an important role in regulating ecdysteroidogenesis in situ at some specific developmental stages. Recent evidence suggested that inactive prothoracic glands from day 0 last instar silkworm larvae are spontaneously activated without stimulation by PTTH. Exogenous PTTH has no or little, if any, effect on either the timing of activation of prothoracic glands in culture or the amount of ecdysteroids secreted (Mizoguchi and Kataoka, 2005). A similar autonomous change in prothoracic gland activity has been well documented in Bombyx last instar larvae (Sakurai and Imokawa, 1988). The present study showed that prothoracic glands from the 1st stages of the last instar (which are inactive in ecdysteroidogenesis) synthesized and secreted the autocrine factor that could stimulate ecdysteroidogenesis by glands from later stages of the last instar larvae (Fig. 5, Gu unpublished data). It may be possible that the autocrine factor released at this stage may play a role in leading to the gradual activation of glands during the later stage. Further study is needed to identify the putative autocrine factor and to explore its relationships with PTTH and in vivo function during insect development.

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In addition, other tissues such as the wing discs or fat body also have stimulatory effects, as determined when prothoracic glands were incubated in wing disc- or fat body-conditioned media (Fig. 10), indicating that these tissues also release a stimulatory factor. However, characteristics of the factor from wing disc-conditioned medium (proteinase K sensitivity and its Mr) differ from the present putative autocrine factor. This result implies that additional paracrine activation of ecdysteroidogenesis of silkworm prothoracic glands may exist. In vertebrate steroidogenic tissues, such as testes, it has been well documented that multiple regulators (endocrine, autocrine, and paracrine) are involved in steroidogenesis in Leydig cells (Saez 1994; Gnessi et al., 1997). In insect steroidogenesis, few studies have been conducted on autocrine and paracrine regulation; more work is needed to clarify this aspect. According to the classical scheme, ecdysteroid synthesis and secretion by prothoracic glands in insects are mainly stimulated by PTTH (Gilbert et al., 2002). Although the identification and characterization of the present putative autocrine factor remain to be carried out, the discovery of the present interesting phenomenon of autocrine activation of silkworm prothoracic glands by a novel factor provides a new perspective on the regulation of ecdysteroidogenesis and raises an important question as to the functional relationships among PTTH, the autocrine factor, and other regulators. In a previous study, an autocrine growth factor which dramatically increased gland cell DNA synthesis in silkworm prothoracic glands was demonstrated (Gu, 2006). The characteristics, such as the molecular weight, heatstability, its lack of retention on C18 Sep-Pak cartridges (Gu, unpublished data), and its resistance to treatment with trypsin and proteinase K, of the present autocrine ecdysiotropic factor, being similar to those of the autocrine growth factor reported in a previous study (Gu, 2006) imply that they may be the same factor. In Drosophila, it has been well documented that the Drosophila insulin-like peptide can activate both cell growth and ecdysteroid synthesis (Brogiolo et al., 2001; Rulifson et al., 2002). In several insect species, it has also been reported that an insulin-like peptide activates ecdysteroidogenesis (Graf et al., 1997). Although it is not clear whether or not the present reported novel autocrine factor belongs to the insulin family, it is interesting to note that the same autocrine factor probably has both growth-promoting and ecdysiotropic activities. Further study is needed to identify this autocrine factor and explore its relationship with PTTH and its in vivo function. In addition, in vitro studies of prothoracic gland activity are generally conducted using short-term incubation and determination of ecdysteroids by RIA. The incubation medium used by researchers usually ranges from 10 to 150 ml. In the present study, the results showed that although no difference in ecdysteroids levels released by silkworm prothoracic glands was observed when using

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between 10 and 50 ml of incubation medium during the first 2 h incubation period, during longer incubation periods (more than 6 h), the volumes of the incubation medium did significantly affect the amount of ecdysteroids produced. The compatibilities of several insect cell culture media were compared for the long-term culture of prothoracic glands from Pseudaletia separate (Komiya et al., 1998). The present finding that decreasing the incubation volume greatly increased ecdysteroid secretion may provide the basis for future research on long-term regulatory factors of prothoracic glands.

Acknowledgements The author thanks the National Science Council for grants NSC94-2313-B-178-001 and 94-2313-B-178-002, and the National Museum of Natural Science of the Republic of China for their financial support.

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