Archives of Insect Biochemistry and Physiology 5:167-177 (1987) Hormonal Control of Ovarian Development in the Silkworm, Bombyx mori Kozo Tsuchida, Masao Nagata, and Akinori Suzuki Departments of Agrobiology (K.T., M.N.) and of Agricultural Chemistry (A.S.),Faculty of Agriculture, University of Tokyo, Bunkyo-Ku, Tokyo, Japan Hormonal control of ovarian development was examined in Bombyx mori. The weight of the ovary increased suddenly by 3 days after pupal ecdysis, and vitellogenin could be immunologically detected in the ovary at that time. The ecdysteroid titers during pupal-adult development, quantified by radioimmunoassay, increased from day 0 to day 2. Ovarian development was arrested for a long period in brainless pupae and isolated pupal abdomens. injection of 20-hydroxyecdysone into such preparations stimulated development of t h e ovaries, and vitellogenin could be detected in ovaries 2 days after injection. The results suggest that 20-hydroxyecdysone acts by stimulating the growth of ovary. Key words: vitellogenin, PTTH, ecdysteroid titer, ovarian development INTRODUCTION The pharate pupa of the female silkworm Bombyx mori L., initiates the production of eggs after somatic growth is completed. The production of mature eggs involves the synthesis of a female-specific protein, vitellogenin, in the fat body and its incorporation into the developing oocyte as vitellin. Silkworm vitellin and vitellogenin have been identified by electrophoretic and immunological techniques [1,2]. Although the vitellogenin may be transported by micropinocytosis [3-51, its uptake into the oocyte is highly selective [61. Hormonal control of oogenesis and vitellogenesis in insects has been extensively reviewed by Hagedorn and Kunkel [7l and Engelmann [S]. In Bombyx and saturniid silkworms, ovarian development occurs during the pupal period and juvenile hormone is not involved, but ecdysteroids have been shown to stimulate ovarian development . Chatani and Ohnishi [lo] reported that exogenous 20-hydroxyecdysone injected into the isolated pupal Received September 4,1985; accepted January 22,1987. Address reprint requests to Dr. Kozo Tsuchida, Department of Biochemistry, Bio Sci West, University of Arizona, Tucson, AZ 85721. 0 1987 Alan R. Liss, Inc. 168 Tsuchida, Nagata, and Suzuki abdomen also induced the initiation of ovarian development in B. mori. In the early half of the pupal stage B. mori vitellogenin is detectable only in the hemolymph, but in the latter half vitellin increases rapidly in the oocyte while vitellogenin levels are maintained in the hemolymph [ll]. This suggests that ecdysteroids are involved in the uptake of vitellogenin into the oocyte during pupal-adult development. Because so little is known about the subject, this paper deals mainly with the relationship of the endocrine system to ovarian development during pupal-adult development of B. mori. MATERIALS AND METHODS Insects The bivoltine race of B. mori, Hosho, was employed. The eggs were incubated at 25°C under LD*18:6, and newly hatched larvae were reared on artificial diet at 25°C under LD 12:12. The pupae were kept at the same conditions as the larval stages. Surgical Techniques Isolated abdomen. Fifth-instar mature larvae started spinning 8 days after the last larval ecdysis and pupated 4 days later. From the third day of the spinning period to the second day after pupation (day 1pupa), each female was ligated between the thorax and abdomen by using a silk thread. The insect was separated at the ligature and the cut surface was sealed with melted wax. The isolated abdomens were kept at 25°C until needed. Brain removal. From day 1 of the spinning period (ie, 3 days before pupation) to within 12 h after pupation, brains were removed from diethylether-anaesthetized specimens. Larval brains were extirpated through a small opening cut in the front of the head capsule. Pupal brains were removed through a small opening cut between the bases of the antennae. A few crystals of phenylthiourea were placed in the hemocoel, and the wound was sealed with melted wax. The brainless pupae were kept at 25°C until needed. Implantation of brain and prothoracic gland. The recipients were anaesthetized-isolated abdomens and brainless pupae that had been prepared as described above. The brains and prothoracic glands of female pharate pupae were removed on day 2 of the spinning period, placed in Grace’s insect saline, and then implanted into the abdominal segment of the anaesthetized host female. Injections Into isolated abdomens 20-hydroxyecdysone (Rhoto Pharmaceutical Co., Osaka, Japan), dissolved in 2% ethanol (0.1, 1.0 or 10 pg/pl), was injected. A *Abbreviations: DW = distilled water; LD = light/dark; PBS = phosphate-buffered saline (NaCI 137 mM, Na2 HP04 3.22 mM, NaH2 PO4 1.28 rnM, pH 7.4); PlTH = prothoracicotropic hormone; RIA = radioimmunoassay. Hormonal Control of Ovarian Development 169 crude extract of PTTH was prepared as described by Suzuki et al. . The crude PTTH from either 2.5 heads or five heads of B. mori adult in 1pl of DW was injected into debrained pupae. The crude extract of B. mori heads contains two species-specific PTTHs, 4K-PTTH is active on Sumiu brainless pupae and 22K-PTTH is active on Bombyx brainless pupae [13,14]. The crude PTTH used in this experiment contained both species (5 unitslhead of 4KPTTH and 1unitlhead of 22K-PTTE-I). Determination of Ovarian Development Ovaries were collected every day from five female pupae. They were rinsed in PBS and blotted on filter paper for several minutes, and their wet weights determined. Quantification of Ecdysteroids The concentration of ecdysteroids in hernolymph was determined by RIA. Antiserum against 20-hydroxyecdysone was prepared by the method of DeReggi et al.  and [23,24-3H]ecdysone (New England Nuclear) was used as the labeled ligand. The antiserum exhibited approximately equal affinity for ecdysone and 20-hydoxyecdysone, but none for cholesterol. The assay was conducted as follows: hernolymph (10 p1) was individually taken from ten insects at 12-h intervals. Ecdysteroids were extracted with 300 pl of methanol; the solvent was evaporated; and the residue was dissolved in 200 pl of DW. Fifty microliters of each sample was mixed with 50 pl of [23,24-3H] ecdysone (about 5,000 cpm), and 100 p1 of antiserum solution, containing I/ 500 the original concentration of the antiserum in borate buffer (0.1 M boric acid, 0.05 M borax, 0.075 M NaCl, pH 8.4) was added. After reaction at 4°C overnight, 200 pl of saturated ammonium sulfate was added and the mixture was centrifuged. The precipitate was washed with 200 pl of 50% saturated ammonium sulfate and then dissolved in 200 pl of DW and mixed with 2 ml of scintillation cocktail (AmSCII, Amersham). Radioactivities were measured for 10 min with a scintillation spectrophotometer; 20-hydroxyecdysone was used as a standard and ecdysteroid amounts were expressed as 20-hydroxyecdysone equivalents. Preparation of Protein Extracts for Electrophoresis and Immunodiffusion Analysis The excised ovaries were homogenized with ten volumes (wlv) of PBS. The ovary suspension was centrifuged at 7,000 g for 15 min at 5°C. Supernatants were stored at -80°C until time of analysis. Immunodiffusion Analysis Double-diffusion tests were performed according to the method of Ouchterlony  in 1.5% agar suspended in Veronal -NaC1 buffer (NaCl 145.3 mM, Veronal 3.1 mM, Veronal sodium 1.8 mM, pH 7.5). Antigen wells were 5 mm from each other. Diffusion was allowed to proceed overnight at 25°C. The gels were then washed thoroughly in PBS. 170 Tsuchida, Nagata, and Suzuki Acrylamide Gel Electrophoresis Slab-electrophoresis of the protein samples were carried out in 7% polyacrylamide gel and 5 mM Tris-glycine buffer at pH 8.3. The gels were stained with 0.5% of Coomassie brilliant blue and destained in 7% acetic acid. RESULTS Eedysteroid Titers The ecdysteroid titers in the hemolymph of B. mori were determined by RIA (Fig. 1).During pupal-adult development these titers changed significantly. They increased dramatically between day 0 (1,600 ngiml hemolymph) and day 2 (2,810 t. 240nglml hemolymph). This result is essentially the same as that obtained with the bioassay technique [17,18]. Ovarian Development of Brainless Pupae Brain removal from female silkworm was performed each day from day 1 of the spinning period until shortly after pupal ecdysis. Almost all of the pharate pupae which had the brain removed by day 2 of the spinning period showed an arrested ovarian development. When the brain was removed on day 3 of the spinning period, ovarian development started after pupation in about 30% of the cases. At 10 days after pupation the mean ovarian weight was 170 mglindividual, while the average weight in unoperated individuals was approximately 500 mg. When the brain was removed within 2 h after pupation, the ovaries began to undergo development in most cases, and by 10 days the mean ovarian weight increased to 210 mg. At 10 days after pupation these brainless individuals showed apolysis and continuing development of the adult integument. 3 lp L z - = 2 E 2 7% p_ f l c s- 3 0 o 1 z 4 5 6 i days a f t s larval-pupal rtdyris 3 i 9 10 Fig. 1. Ecdysteroid titer (ng 20-hydroxyecdysone equivalent/ml) in the hemolymph during female pupal-adult development. The data represent the mean f SD for ten animals at each time point. Hormonal Control of Ovarian Development 171 Three brains that had been removed from pharate pupae on day 2 of the spinning stage were implanted into the abdomens of debrained pupae on 10 days after surgeon. The recipients developed and the weight of their ovaries increased to a mean of ca 120 mg on the 10th day after implantation (Fig. 2). Crude PTTH was injected into pupae that were in a state of arrested ovarian development as a result of brain removal. As described in the Materials and Methods, the crude PTTH injection was carried out by using two dosage levels: 2.5 headslF1 DW and 5 headslpl DW. The weight of ovaries of the injected pupae started to increase at about 3 days after injection (Fig. 3). In the case of the five-heads dose, the mean ovarian weight was about 100 mglpupa 10 days after injection. In the 2.5-heads dose, the ovaries developed only to a mean weight of 35 mglpupa. Accordingly, it is apparent that the /" P P L .m a P m y. i 50 z: 0 2 4 6 8 m y s after implantation 10 Fig. 2. The effects of implanting of three brains ( V ) or one brain (B) on ovarian development in debrained female pupa. The open squares show the ovarian development of brainless pupae. m *- 0 0 2 4 6 8 Days after injection 10 Fig. 3. Ovarian development obtained after injecting crude P T H extracts into brainless abdomens of female pupae (CI,5 headslpl; V,2.5 heads/pI; 0 ,control). 172 Tsuchida, Nagata, and Suzuki injection of crude P’ITH induces an ovarian development the degree of which is dependent upon the dose injected. Ovarian Development in Isolated Abdomens Isolated abdomens were prepared by performing thorax-abdomen ligatures from day 3 of the larval spinning period to day 2 of the pupal stage. In the case of ligatures performed up to day 0 of the pupal stage, no signs of ovarian development were seen during the entire observation period (about 30 days). However, 70% of the isolated pupal abdomens ligatured on the first day after pupation initiated ovarian development, and the ovarian weight increased up to about 310 mglfemale by 10 days postpupation. In addition, when the ligature was performed on day 2 of the pupal stage, almost all of ovaries in the isolated abdomens showed growth, and continued to increase in ovarian weight to a mean value of approximately 380 mglindividual on 10 days after pupation. Isolated abdomens prepared by ligaturing within 12 h after pupation were stored for 10 days at 25°C. Subsequently, three brains from day 2 spinningstage larvae were implanted into each isolated abdomen. The ovaries of the isolated abdomens with implanted brains did not develop; the mean ovarian weight was only 20 mglpupa, even on the 10th day after implantation. However, when three brains and three prothoracic glands were implanted, the weight of the ovaries increased, reaching a value of about 135 mglfemale at 10 days after implantation (Fig. 4). Isolated abdomens prepared by ligaturing within 12 h after pupation were stored for 10 days and then injected with 1pl of solution containing 0.1, 1.0, or 10 pg of 20-hydroxyecdysone. Regardless of the injected dose, the weight of ovaries began to increase 2 days after injection. The development of ovaries was positively correlated with the amount of hormone injected; at 10 days after injection the ovarian weight was 75 mglfemale in the 0.1-pg group, 120 mg in the 1.0-pg group, and 210 mg in the 10-pg group (Fig. 5). 2 4 6 8 m Days after implantation Fig. 4. Ovarian development in isolated abdomens of female pupae after implanting three brains and three prothoracic glands ( 0 ), or three brains ( 0 ). The open triangles indicate control values. Hormonal Control of Ovarian Development 0 2 4 6 8 173 10 Days after injection Fig. 5. Ovarian development obtained after injecting varying doses of 20-hydroxyecdysone (0 ; 10 pg/ul, 0 ;1 pg/pI, V ;0.1 pglpl) into isolated abdomens of female pupa. When I pg/pl of the juvenile hormone mimic ZR515 was injected (El), the ovary showed almost no development. In contrast to these results, when 1pg/pl acetone of the juvenile hormone mimic ZR515 was injected into each isolated abdomen, the ovaries showed almost no development. Effect of Crude PTTH and 20-Hydroxyecdysone on Ovarian Protein The brain was removed on day 2 of the spinning stage. At 10 days after this procedure, the debrained pupae were injected with crude PTTH (5 headslpl DW) or 20-hydroxyecdysone (1 pglpl 2% ethanol). Changes in ovarian proteins after injections were monitored by polyacrylamide gel electrophoresis. In the case of the debrained pupae injected with the crude PTTH, the amount of ovarian protein increased at 4 days after injection. In regard to pupae injected with 20-hydroxyecdysone, the electrophoresis pattern was found to change more quickly than in pupae injected with PTTH (Fig. 6 ) . Hormonal Control of Incorporation of Vitellogenin Into the Ovaries By using Ouchterlony’s agar-gel immunodiffusion , the relationship between the ovarian incorporation of vitellogenin and 20-hydroxyecdysone was investigated. In debrained pupae, no incorporation of vitellogenin was detected even after several weeks. However, when crude MTH (5 headslpl DW) or 20-hydroxyecdysone (1pg/pl2% ethanol) was injected into debrained pupae, vitellogenin could be detected in the ovaries 2 or 3 days after injection (Fig. 7). DISCUSSION In many insects, ovarian development is controlled by hormone(s) [S], and juvenile hormone seems to be the primary hormone. However, in the case 174 Tsuchida, Nagata, and Suzuki Days after injection 0 2 4 6 8 1012 0 2 4 6 8 1012 anode 2 O - h ~ r o x ~ ~ y ~ ~ crude PT 1 H Fig. 6. The effect of crude PTTH and 20-hydroxyecdysone on ovarian proteins of debrained pupa. Pupae (ten days after debraining)were injected with crude PTTH or 20-hydroxyecdysone. PTTH I 0 2 4 681012 Days after injection Fig. 7. Ouchterlony's immunodiffusion with ovary extract. Center well held vitellogenin antiserum and both side wells hold ovary extract from debrained pupae. When 5 headslpl crude PTTH extract (above) or 1 pglpl 20-hydroxyecdysone (below) were injected into the debrained pupae, vitellogenin could be detected in the ovary 2 days after injection. Numbers in parentheses show the days after debraining. Hormonal Control of Ovarian Development 175 of the silkworm and other Lepidoptera, ecdysteroids have been reported to play a central role in oQariandevelopment [9,18]. The present study revealed the following points: (1)In B. mori, the brain secretes PTTH before pupation and thereby stimulates the prothoracic gland. (2) The prothoracic gland secretes ecdysone; its concentration in the hemolymph reached a peak (ca 3 pglml) on day 2 of the pupal stage and then decreased after day 4 to reach a low point (ca 5 nglml) on day 6 of the pupal stage. (3) The weight of the ovary increases very suddenly after day 3 of the pupal stage, and vitellin can be immunologically detected in the ovary on this day. It is known that if B. mori and other species of Lepidoptera are debrained at a certain time during their development, adult differentiation is arrested, and they become so-called "Dauer pupae" [19-211. According to Ishizaki , the time of the critical period of MTH secretion for adult development varies markedly among the races of B. mori, ranging from the feeding period of the fifth-instar larva to shortly after larval-pupal ecdysis. In this study, the Chinese race "Hosho" was employed, and if this race is debrained before pupation, its adult development is totally arrested. In addition, if isolated abdomens are prepared from the silkworm before pupation, adult development does not occur. Although ovarian development is arrested for a long time in these debrained pupae and isolated abdomens, injection of 20-hydroxyecdysone enables the ovaries to develop. Moreover, the weight of the ovary increases in direct proportion to the dose of 20-hydroxyecdysone injected. This phenomenon is also related to an increase in number of mature follicles; when the injected dose of 20-hydroxyecdysone was small, approximately half of the follicles in the ovariole were found to be absorbed. The same results have been reported by Chatani and Ohnishi [lo], who also observed that exogenous ecdysteroids are immediately inactivated following injection into isolated abdomens, with no detectable activity on the second day after the injection. On the basis of these findings it can be concluded that ecdysteroids are essential for ovarian development in B. moui, and they need to be secreted continously if ovarian development is to be completed. This idea is supported by the ecdysteroid titers determined by RIA reported here and elsewhere [El. After the peak level of the ecdysteroids is achieved in the hemolymph, the development of the ovary begins. At the same time, vitellogenin becomes immunologically detectable in the ovary. Vitellogenin could not be found in the ovaries of debrained pupae or isolated abdomens even 10 days after operation, but there is immunological evidence for its presence in the ovary on the second day after injection. This may mean that 20-hydroxyecdysone acts on the follicular epithelial cells of the ovary to make them competent for vitellogenin uptake. In Drosophilu  and Sarcophuga  it is known that ecdysteroids stimulate the vitellogenin synthesis. Izumi and Tomino  reported that the biosynthesis of vitellogenin in B. mori starts immediately after pupation, and Ohno et al.  showed that ecdysteroid could stimulate the biosynthesis of hemolymph proteins including female specific protein. 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