Local expression and distribution of a storage protein in the ovary of Hyphantria cunea.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 48:111–120 (2001) Local Expression and Distribution of a Storage Protein in the Ovary of Hyphantria cunea Hyang-Mi Cheon,1 Hong-Ja Kim,1 Duck-Hwa Chung,2 Myeong-Ok Kim,1 Joong-Suk Park,1 Chi-Young Yun,3 and Sook-Jae Seo1* 1 2 Division of Life Science, College of Natural Sciences, Gyeongsang National University, Chinju, Korea Division of Applied Chemistry and Food Science and Technology, College of Agriculture, Gyeongsang National University, Chinju, Korea 3 Department of Biology, Taejon University, Taejon, Korea Storage protein-1 (HcSP-1) is a major storage protein found in the hemolymph and fat body of Hyphantria cunea. HcSP-1 has a high methionine (6.0%) and low aromatic amino acid content (8.5%) (Cheon et al., 1998). In this study, the accumulation and expression of HcSP-1 in ovary was investigated using biochemical and immunocytochemical methods. HcSP-1 was detected in the ovaries in 6-day-old pupae and accumulated toward the end of pupal life, when HcSP-1 transcripts were detectable by Northern blot analysis and RT-PCR. In situ hybridization showed that the HcSP-1 mRNA was located in the nurse cells and follicular epithelial cells, but not in the oocyte. Though most of the HcSP-1 that is incorporated in the yolk bodies of the oocyte is probably sequestered from the surrounding hemolymph, HcSP-1 is an important yolk protein contributing to early yolk body formation before the development of patency by the follicular epithelium. Arch. Insect Biochem. Physiol. 48:111–120, 2001. © 2001 Wiley-Liss, Inc. Key words: local expression; storage protein; ovary; Hyphantria cunea INTRODUCTION In holometabolous insects, storage proteins represent a major protein component of the larval hemolymph (Wyatt and Pan, 1978; Levenbook, 1985; Roberts and Brock, 1981). These proteins are synthesized in large quantities by the fat body of actively feeding larvae and are released into the hemolymph. Other larval cell types including the midgut (Palli and Locke, 1987a), epidermis (Palli and Locke, 1987b), pericardial cells (Fife et al., 1987), and testis (Miller et al., 1990) also synthesize and export storage proteins, but their proportional contribution to the total hemolymph complement is probably rather small. The larval hemolymph of the fall webworm, © 2001 Wiley-Liss, Inc. Hyphantria cunea accumulates two forms of storage protein termed HcSP-1 and HcSP-2 (Kim et al., 1989). HcSP-1 has a relatively high methionine (6.0%) and a low aromatic amino acid content (8.5%) (Cheon et al., 1998), but does not Contract grant sponsor: Brain Korea 21 project. Abbreviations used: RT-PCR = reverse transcription-polymerase chain reaction; SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis; SP = storage protein. *Correspondence to: Sook-Jae Seo, Division of Life Science, College of Natural Sciences, Gyeongsang National University, Chinju, 660-701, Korea. E-mail: email@example.com Received 24 October 2000; Accepted 4 June 2001 112 Cheon et al. exhibit sexual dimorphism (Kim et al., 1989, Song et al., 1997). It has been suggested that high-methionine hexamers of lepidopterans play a role in egg formation because of their greater abundance in females than males (Bean and Silhacek, 1989; Ryan et al., 1985; Tojo et al., 1980). The synthesis or expression of a storage protein in the ovary has not been previously reported. Therefore, the goal of the present study was to use Northern blot hybridization, RT-PCR, Southern blot, and in situ hybridization to determine whether the HcSP-1 gene is locally expressed in the ovary. Finally, we considered the relationship between oogenesis and locally expressed SP-1 in H. cunea. MATERIALS AND METHODS Animals Fall webworms, Hyphantria cunea, were reared on artificial diet at 27°C and 75% relative humidity with a photoperiod of 16 h light:8 h dark. Ovary Preparation Ovaries were dissected from 6-, 8-, and 10day-old female pupae and adults, and were cleaned of attached fat body cells. It was not practical to analyse ovaries from stages earlier than 6-day-old pupae. The samples were homogenized in Ringer’s solution (150 mM NaCl, 1.8 mM CaCl2, 1.3 mM KCl, 10 mM Tris, pH 7.0) using a Dounce glass-Teflon homogenizer (Seo et al., 1998). The homogenates were centrifuged at 10,000g for 30 min at 4°C. The supernatant was removed and stored at –70°C. Electrophoresis SDS-PAGE of ovarian samples was performed according to the method of Laemmli (1970) using a 12.5% separating slab gel. All samples were heated at 90°C for 9 min in the presence of 2% SDS and 5% 2-mercaptoethanol. Gels were stained with Coomassie blue following completion of electrophoresis. Western Blot Following SDS-PAGE, proteins in the gel were electrotransferred to a sheet of nitrocellulose (0.45 µm, Bio-Rad, Hercules, CA) according to the procedure of Towbin et al. (1979). The blots were blocked in 20 mM Tris-HCl pH 7.6, 137 mM NaCl, and 0.2% Tween-20 (buffer A) containing 5% nonfat dry milk, and then incubated with antiserum against HcSP-1 (Seo et al., 1998) at a 1:1,500 dilution in buffer A. After washing in buffer A, the blots were incubated with horseradish-peroxidase-conjugated goat anti-rabbit IgG (1:3,000) in buffer A for 1 h. Immunoreactivity was determined using the enhanced chemiluminescence (ECL) reaction (Amersham, Buckinghamshire, UK). RNA Isolation, RT-PCR, and Southern Blot Analysis The temporal profiles of transcript abundance for the storage protein in ovaries from 6-, 8-, and 10-day-old pupae and adult were examined using RT-PCR followed by Southern blotting with specific radioactive probes. Total RNA was isolated from ovary and other organs by lysis buffer, spin column, and wash buffer according to the protocol recommended by the manufacturer (Qiagen Inc. Chatsworth, CA). Five-microgram aliquots of each RNA preparation were reversetranscribed by the Superscript II reverse transcriptase (Gibco BRL) using Oligo(dT)12–18 primers (Gibco BRL) in a reaction volume of 25 µl. The reverse transcription products were diluted to 50 µl with TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0) and stored at –20°C as a cDNA pool until use. For developmental profile analysis, 0.5 µl ovary equivalents from the cDNA pools were used as PCR templates. A 539-bp specific fragment was amplified with the forward primer, 5′-CTTCGGCCAGCGTCGTCAA-3′, and the reverse primer, 5′-TGCGGCTCTGGTCATTTTCATC-3′. Thermal cycling conditions were as follows: the reaction was incubated at 94°C for 5 min followed by 35 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 60 s. After PCR amplification, 10 µl each of the total 50 µl reaction was fractionated on a 1.2% agarose gel and transferred onto a Hybond N+ membrane (Amersham) under alkaline conditions. Radioactive cDNA probes were prepared from 25 ng HcSP-1 cDNA (EcoRV digested 1.5-kb cDNA fragment). The HcSP-1 cDNA fragments were labeled by a random-primer DNA labeling system (Gibco BRL) to incorporate [α-32P]dATP (NEN). The membrane was prehybridized with 1.5 × SSPE (20 × SSPE: 3 M NaCl, 0.2 M NaH2PO4, 0.02 M EDTA), 7% sodium dodecylsulfate (SDS), Expression of Storage Protein in H. cunea Ovary 10% polyethylene glycol (PEG), 0.25 mg/ml bovine serum albumin (BSA), and 0.1 mg/ml denatured salmon sperm DNA (Gibco BRL) for 4 h at 65°C. Hybridization was performed for 18 h at 65°C in the prehybridization buffer with 5 × 105 cpm/ml 32P-labelled probes. The membrane was washed twice at 65°C in 2 × SSC (10 × SSC: 1.5 M NaCl, 0.15 M sodium citrate), 0.1% SDS for 15 min, twice in 0.1 × SSC, 0.1% SDS for 15 min, and then autoradiographed. Southern blotting of the PCR-amplified total RNA preparations without reverse transcription did not result in any appreciable signals, indicating that contamination of RNA preparations with genomic DNA fragments was negligible (not shown). Northern Blotting Ten and thirty micrograms of total RNA from fat body and other tissues, respectively, were denatured and subjected to electrophoresis in a 1.2% agarose gel containing 2.2 M formaldehyde. Following electrophoresis, gels were rinsed in 10 × SSC and transferred to nitrocellulose (Schleicher and Schuell) in 10 × SSC. Blots were prehybridized with 1.5 × SSPE, 7% SDS, 10% PEG, 0.1 mg/ml sonicated denatured salmon sperm DNA, and 0.25mg/ml BSA for 4 h at 65°. Hybridization was performed for 18 h at 65°C in the prehybridization buffer with 5 × 105 cpm/ml 32P-labelled probes prepared according to the method of random priming (Feinberg and Vogelstein, 1983). The filter was washed twice with 1 × SSC, 0.1% SDS at 65°C for 15 min, and twice subsequently for 15 min with 0.1 × SSC and 0.1% SDS at 65°C before exposure to X-ray film at –70°C. In Situ Hybridization In situ hybridization was performed based on the protocol reported by Petraglia et al. (1992). Fixed ovary tissues were sectioned at 10 µm with a cryostat at –20°C. The tissue sections were hybridized with a 35S-labelled HcSP-1 cRNA probe for 1 day at 60°C in a humid chamber. Sections were incubated with RNase A to exclude the possibility of mis-matched sequences. After washing and drying, sections were apposed to NTB2 emulsion (Eastman Kodak) for 2 weeks. The signals were observed under a microscope. The control section was pretreated with RNase and showed no signal. 113 Immunocytochemistry Immunocytochemistry was performed as previously described (Miller et al., 1990). Ovaries were fixed for 3 h in a mixture of 4% formaldehyde and 1% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.5) containing 0.15 mM CaCl2 and 0.45 M sucrose (FM). Fixation was completed by incubating the ovaries overnight in pH 10.4 FM without glutaraldehyde. The tissues were rinsed in 0.1 M sodium phosphate buffer (pH 7.5), dehydrated in a graded ethanol series (up to 95%), and embedded in Lowicryl K4M (Polysciences, Warrington, PA). Ultrathin sections mounted on formvar-coated nickel grids were treated for 10 min with Tris-buffered saline (TBS; 0.02 M Tris-HCl, pH 7.5 containing 0.5 M NaCl). The sections were etched with 3% H2O2 in double distilled H2O for 5 min and then blocked with 3% BSA in TBS for 30 min. The sections were incubated with 1:200 diluted antiserum against HcSP1 in TBS plus 1% Tween-20 (TBS/Tween) for 60 min. Following a wash in TBS/Tween 3 times for 15 min with gentle agitation, the sections were exposed to gold-goat anti-rabbit IgG (20 nm: Zymed, San Francisco, CA) diluted 1:5 in TBS/ Tween for 60 min. The grids were washed with 0.3% BSA in TBS, and the sections poststained with 2% uranyl acetate followed by 0.2% Reynolds’ lead citrate (Reynolds, 1963). Ultrastructural examination was performed on a Hitachi H-600 transmission electron microscope operating at 75kV. Controls included: (1) substitution of preimmune serum for primary antiserum; (2) use of secondary antibody in the absence of treatment with primary antibody; and (3) treatment of thin sections with colloidal gold alone. RESULTS Accumulation of SP-1 in the Ovary Ovary extracts from female pupae and adult were compared by SDS-PAGE and Coomassie blue staining to determine quantitative changes in the concentration of HcSP-1 (Fig. 1). HcSP-1 was detected in small amounts in the ovaries in 6-dayold pupae, and had accumulated in large amounts in pupae by day 10. The accumulation of HcSP-1 in the ovary toward the end of the pupal stage coincides with a slight decline in the hemolymph, suggesting a redistribution of SP-1 from hemo- 114 Cheon et al. Locke, 1987a), epidermis (Palli and Locke, 1987b), and testis (Miller et al., 1990) also synthesize and export arylphorin. We, therefore, decided to determine if the HcSP-1 gene is expressed in the ovary. Accordingly, ovaries were dissected from day-6 pupae to adult and were thoroughly cleaned of all visible adhering fat body. Total RNA from fat body, midgut, ovary, and testis were hybridized with HcSP-1 cDNA probe. Northern blot analyses indicated that no transcript was present in midgut, but that a substantial amount was present in fat body, and a lesser amount in the gonad (Fig. 2A). The HcSP-1 probe revealed that the same 2.5-kb transcript accumulated in the ovary as in the fat body. Developmental Pattern of the HcSP-1 Transcript in Ovary and Fat Body Fig. 1. SDS-PAGE of ovary extracts of female H. cunea. Each lane was loaded with 20 µg of ovary proteins. Bottom: Immunoblot analysis probed with HcSP-1 antiserum. Molecular weight standards (× 103) and three yolk proteins are marked on the right (YP1 from Lee et al., 1988; YP2 from Lee et al., 1995; YP3 from Lee and Kim, 1991). HcSP1 is marked on the left. P6-P10, 6-, 8-, and 10-day-old pupae; A0, newly eclosed adult. lymph into the ovary or other adult tissues (Seo et al., 1998). However, there was an abrupt decrease in HcSP-1 content in the ovaries after adult emergence (Fig. 1). In a double immunodiffusion test, antiserum against HcSP-1 showed very weak precipitation lines with an extract of adult ovaries (Lee et al., 1990). HcSP-1 is probably present in the ovaries of pupae less than 6 days old, but they were too small to sample for biochemical analysis. It is noteworthy that ovaries selectively accumulated several polypeptides, including three kinds of yolk proteins (Fig. 1). Yolk proteins were accumulated in larger quantities by the end of the pupal stage and in adults. Distribution of SP-1 Transcript Among Organs Ray et al. (1987) argued that the arylphorin gene is expressed exclusively in fat body cells of wax moth larvae. However, in some insects, other larval cell types such as the midgut (Palli and The developmental profile of the HcSP-1 transcript in H. cunea ovary could not be determined by Northern blot, because the HcSP-1 transcript was expressed at low levels. Therefore, we performed RT-PCR analysis using HcSP-1 genespecific primers (Fig. 2B). 0.6-kb PCR products were detected in ovary from all developmental stages tested except adults. Adult ovaries appear to be in the equivalent of a post-vitellogenic stage, when follicles show no remaining nurse cell cap indicating the termination of vitellogenesis (Zimowska et al., 1991). The developmental profiles of HcSP-1 and HcSP-2 transcripts, as determined by Northern blot, were markedly different in female fat body (Fig. 2C). In general, the relative abundance of HcSP-1 transcript in the female fat body is considerably greater than that of the HcSP-2 transcript. The quantity of HcSP-1 transcript was greatest in late 7th instar larvae, at a time when HcSP-2 transcript was present only at a trace level. HcSP-1 levels gradually declined thereafter and remained low throughout pupal development (Fig. 2C, SP-1). In contrast, the HcSP-2 transcript was present at trace levels before the prepupal stage, reached its peak during prepupal and early pupal stages, and then drastically declined after 2-day-old pupae (Fig. 2C, SP-2). In contrast, no big differences in developmental profiles between the two storage protein transcripts were observed in males (data not shown). Expression of Storage Protein in H. cunea Ovary 115 Fig. 2. Presence of HcSP-1 transcript among tissue types revealed by Northern blot (A). Developmental accumulation of the HcSP-1 transcript from ovary (B) and fat body (C) revealed by RT-PCR (B) and Northern blot (C), respectively. Total RNA from fat body (10 µg) and other tissues (30 µg) were separated and probed. For further details see Materials and Methods. Fb, fat body; Mg, midgut; Ov, ovary; Te, testis; 7E-L, early, middle, and late 7th instar larvae; PP, prepupae; P0-P10, pupae at days 0–10 of development; A0, newly eclosed adult. In Situ Hybridization and Localization of the HcSP-1 Transcript in the Ovary between the follicular epithelial cells surrounding the oocyte, but true patency was not yet evident. At this stage, a fair amount of HcSP-1 was associated with the tunica propria and a few gold particles labeling SP-1 were present in structures that seemed to be the Golgi complex (Fig. 4A), but the labeling was almost at background level. The small yolk spheres containing HcSP-1 were detectable at the periphery of the oocyte (Fig. 4B), while the section labeled with antisera to YP2 showed a few gold particles in yolk spheres (Fig. 4C). Two antisera against YP1 and YP3 showed no labeling in the same section (data not shown). During the vitellogenic stage, follicular epithelial cells achieve patency, with large interfollicular spaces developing at the apical surfaces (Seo et al., 1998). Once detected in the perioocytic space, HcSP-1 was also detected within the transitional yolk bodies. Smaller transitional yolk bodies appeared to fuse with one another to form The precise cellular localization of HcSP-1 mRNA in the ovary was examined by in situ hybridization. HcSP-1 specific labeling was observed in the nurse cells (Fig. 3A) and the follicular epithelial cells (Fig. 3C–E) surrounding the oocyte, but not in the cytoplasm of the oocyte where the protein granules were packed (Fig. 4). In most follicles from the adult stage (Fig. 3F), the follicular epithelial cells were engaged in prechoriogenic activity and the nurse cell cap had disintegrated (data not shown). At this stage, most follicular epithelial cells showed no hybridization signal, but a few cells still gave weak signals (Fig. 3F). Immunocytochemistry Just before vitellogenic (provitellogenic) ovaries, small interfollicular spaces were observed 116 Cheon et al. Figure 3. Expression of Storage Protein in H. cunea Ovary 117 Fig. 4. The localization of HcSP-1 associated the early yolk body formation in provitellogenic ovary of H. cunea. Note that HcSP-1 label is present on the tunica propria (Tp) in considerable amounts. The follicular epithelium has small intercellular spaces between adjacent plasma membranes (A). Early yolk bodies containing HcSP-1 are observed around the periphery of the oocyte (B). Immunogold labeling for HcSP-1 (B) is stronger than the minimal labeling for YP2 (C). Magnification bar = 0.5 µm. large mature yolk bodies that were heavily labeled with colloidal gold (Fig. 5). hemolymph, and sequestered again by the fat body at the end of the feeding period (Kim et al., 1989). Storage protein can be used during metamorphosis and oogenesis as a source of amino acids (Tojo et al., 1980; Karpells et al., 1990). In B. mori, the transcript and the protein are expressed in both male and female fat bodies during the fourth instar, but they are restricted to females during the fifth instar (Izumi et al., 1988; Sakurai et al., 1988). Ogawa and Tojo (1981) suggested that the female-specific SP-1 of B. mori may supply amino acids for the formation of the egg yolk protein precursor vitellogenin (Engelmann, 1979). The relatively high methionine content of the B. mori SP-1 may be metabolized to cystine for chorion formation (Inokuchi, 1972; Sumioka and DISCUSSION SP-1 of Hyphantria cunea is a storage protein produced by the fat body, exported to the Fig. 3. Localization of HcSP-1 mRNA in the ovarian follicle by in situ hybridization using a 35S-end labeled cRNA fragment of HcSP-1. Hybridization signals (black dots) indicative of HcSP-1 mRNA are visible in the nurse cells (A,C,E) and follicular epithelial cells (A,C,D,E,F). No signal was identified in the control section pretreated with RNase (B). A: Previtellogenic follicle. C: Early vitellogenic follicle. D: Cross-section of oocyte in early vitellogenic stage, E,F: Post-vitellogenic follicle. Nc, nurse cell; Fc, follicular epithelial cell; Oc, oocyte. 118 Cheon et al. Fig. 5. The HcSP-1 internalization pathway in the H. cunea oocyte during the vitellogenic stage. Once in the perioocytic space (Ps), the storage protein (dark spots) is incorporated into the transitional yolk body (*). Subsequently, the transi- tional yolk body, which contains HcSP-1, is transformed into a mature yolk body (4). Numbers 1–4 show the transition from immature yolk body to mature yolk body. Ld, lipid droplet. Scale bar = 1 µm. Yoshitake, 1974). SP-1 in Hyphantria cunea has a somewhat higher content of methionine (6.0%), indicating the possibility of contribution to egg formation. Larval hemolymph protein synthesis in lepidopterans occurs primarily, but not exclusively, in larval fat body cells (Kanost et al., 1990). In this report, we present evidence for storage protein transcription in the ovary of Hyphantria cunea. However, the level of the HcSP-1 transcript was much lower in the ovary than in fat body cells. The size of the HcSP-1 transcript appeared to be identical in the two cell types (Fig. 2A), suggesting that there are no tissue-specific differences associated with HcSP-1 RNA processing. Presence in the follicle of a protein transcript considered up to now to be a storage protein is rather unexpected. The persistence of the HcSP-1 transcript in the ovary of late pupae is similar to a phenomenon in Heliothis virescens where the testes cells continue to transcribe the arylphorin gene in the pharate adult and adult stages (Miller et al., 1990). During the early stages of follicle growth, HcSP-1 transcript is expected to present in follicles, but it is almost impossible to identify it by biochemical methods because of the minuteness of the follicles. Just before the vitellogenic stage, HcSP-1 is already present in small yolk bodies around periphery of the oocyte. This phenomenon is very similar to the incorporation of YP2 into the early yolk bodies during the provitellogenic stage of Plodia interpunctella (Zimowska et al., 1994), in which the interfollicular spaces are present but are not yet truly patent (Zimowska et al., 1994). The labeled HcSP-1 in the early yolk bodies seems to be originated from follicular epithelial cells rather than from nurse cells. Though HcSP-1 transcripts are observed in the nurse cell of H. cunea ovary, it is very difficult to have evidence for the transport of HcSP-1 mRNA or protein itself from nurse cells to oocyte. Even HcSP-1 was distributed randomly in the cytoplasm of nurse cells, it was not possible to determine whether Expression of Storage Protein in H. cunea Ovary the HcSP-1 in the nurse cells was from hemolymph or from de novo synthesis (Seo et al., 1998). Immunocytochemical method was not sufficient to make any identification of biosynthetic activity in the nurse cells. Though the labeling of HcSP-1 in early yolk bodies is not intense, its contribution to the early yolk bodies is much greater than that of yolk protein. The HcSP-1 transcript is found in 6- through 10-day-old pupae, so HcSP-1 synthesis is presumably continuous from the previtellogenic to vitellogenic stage of H. cunea. Since t he immunogold labeling of three Yps is not above background levels in early yolk bodies, their contribution to early yolk bodies is apparently rather small. During the vitellogenic stage, large protein granules densely labeled with HcSP-1 were observed throughout the oocyte. At this stage, the follicular epithelial cells exhibit patency with large interfollicular spaces and no cell contacts at the apical surfaces, permitting transport of material in the hemolymph to the surface of the oocyte (Seo et al., 1998). We observed that the H. cunea storage protein is actively taken up into the protein granules by the developing oocyte and serves as a yolk protein during egg formation (Seo et al., 1998). Though the incorporation of ovarian HcSP-1 into yolk bodies is smaller than that of the hemolymphatic HcSP-1, this transcript in follicles might contribute to the formation of early yolk bodies before the vitellogenic stage. ACKNOWLEDGMENTS We are very grateful to Dr. Thomas W. Sappington (USDA-ARS, IFNRRU) for a critical reading of the manuscript and to Dr. Seol Kwang Youl (National Sericulture and Entomology Research Institute, RDA) for the fall webworm supply. LITERATURE CITED Bean DW, Silhacek DL. 1989. Changes in the titer of the female-predominant storage protein (81K) during larval and pupal development of the waxmoth, Galleria mellonela. Arch Insect Biochem Physiol 10:333–348. Cheon HM, Hwang IH, Chung DH, Seo SJ. 1998. Sequence analysis and expression of Met-rich storage protein SP-1 of Hyphantria cunea. Mol Cells 8:219–225. 119 Engelmann F. 1979. Insect vitellogenin: identification, biosynthesis, and role in vitellogenesis. Adv Insect Physiol 14:49–108. Feinberg AO, Vogelstein B. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13. Fife HG, Palli SR, Locke M. 1987. A function for pericardial cells in an insect. Insect Biochem 17:829–840. Inokuchi TC. 1972. Isolation and determination of lanthionine and its metabolism in the silkworm Bombyx mori L. Bull Sericul Exp Sta 25:169–197. Izumi S, Sakurai H, Fuji T, Ikeda W, Tomino S. 1988. Cloning of mRNA sequence coding for sex-specific storage protein of Bombyx mori. Biochem Biophys Acta 949: 181–188. Kanost MR, Kawooya JK, Ryan RD, Van Heusden MC, Ziegler R. 1990. Insect hemolymph proteins. Adv Insect Physiol 22:299–366. Karpells S, Leonard D, Kunkel J. 1990. Cyclic fluctuations in arylphorin, the principal serum storage protein of Lymantria dispar indicate multiple roles in development. Insect Biochem 20:73–82. Kim HR, Kang CS, Mayer RT. 1989. Storage proteins of the fall webworm, Hyphantria cunea Drury. Arch Insect Biochem Physiol 10:115–130. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. Lee SD, Seo EW, Kim HR. 1988. Major yolk proteins of Hyphantria cunea Drury. Korean J Entomol 18:65–72. Lee SD, Kim HR. 1991. Synthesis and fate of yolk protein-3 in Hyphantria cunea Drury. Korean J Zool 34:394–402. Lee SD, Lee SS, Kim HR. 1995. Purification and characterization of yolk protein-2 from the fall webworm, Hyphantria cunea Drury. Arch Insect Biochem Physiol 28:113–129. Lee SN, Kang CS, Kim HR. 1990. Fate and distribution of storage protein-1 in Hyphantria cunea Drury. Korean J Entomol 20:33–40. Levenbook L. 1985. Insect storage protein. In: Kerkut GA, Gillbert LI, editors. Comprehensive insect physiology, biochemistry and pharmacology. New York: Pergamon Press; p 307–346. Miller SG, Leclerc RF, Seo SJ, Malone C. 1990. Synthesis and transport of storage proteins by testes in Heliothis virescens. Arch Insect Biochem Physiol 14:151–170. Ogawa K, Tojo S. 1981. Quantitative changes of storage proteins and vitellogenin during the pupal-adult development in the silkworm, Bombyx mori. Lepidoptera : Bombycidae. Appl Ent Zool 16:288–296. 120 Cheon et al. Palli SR, Locke M. 1987a. Hemolymph protein synthesis by the larval midgut of an insect, Calpodes ethlius (Lepidoptera, Hesperiidae). Insect Biochem 17:561–572. Palli SR, Locke M. 1987b. The synthesis of hemolymph proteins by the larval epidermis of an insect Calpodes ethlius (Lepidoptera : Hesperiidae). Insect Biochem 17:711–722. Petraglia F, Woodruff TK, Botticelli G, Botticelli A, Genazzani AR, Mayo KE, Vale W. 1992. Gonadotropin-releasing hormone inhibin and activin in human placenta; Evidence for a common cellular localization. J Clin Endocrinol Metabol 74:1184–1188. Ray A, Memmel N, Kumaran A. 1987. Developmental regulation of the larval hemolymph protein genes in Galleria melonella. Wilhelm Roux’s Arch Dev Biol 196: 414–420. Reynolds ES. 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 17:208–212. Roberts D, Brock H. 1981. The major serum proteins of Dipteran larvae. Experientia 37:103–110. Ryan RO, Keim PS, Wells MA, Law JH. 1985. Purification and properties of a predominantly female-specific protein from the hemolymph of the larvae of the tobacco hornworm Manduca sexta. J Biol Chem 260:782–787. Sakurai H, Fuji T, Izumi S, Tomino S. 1988. Structure and expression of gene coding for sex-specific storage protein of Bombyx mori. J Biol Chem 263:7876–7880. Seo SJ, Kang YJ, Cheon HM, Kim HR. 1998. Distribution and accumulation of storage protein-1 in ovary of Hyphantria cunea Drury. Arch Insect Biochem Physiol 37:115–128. Song JK, Nha JH, Kim HR. 1997. Comparative analysis of storage proteins of the fall webworm (Hyphantria cunea Drury). Comp Biochem Physiol 118B:123–129. Sumioka H, Yoshitake N. 1974. Variation of free amino acids in various tissues of the silkworm during the oogenesis. J Sericul Sci Japan 43:65–71. Tojo S, Nagata M, Kobayashi M. 1980. Storage proteins in the silkworm Bombyx mori. Insect Biochem 10:289–303. Towbin H, Staehelin T, Gordon J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354. Wyatt GR, Pan ML. 1978. Insect plasma proteins. Ann Rev Biochem 47:779–817. Zimowska G, Silhacek DL, Shaaya E, Shirk PD. 1991. Immunofluorescent analysis of follicular growth and development in whole ovaries of the Indian meal moth. J Morphol 209:215–228. Zimowska G, Shirk PD, Silhacek DL, Shaaya E. 1994. Yolk sphere formation is initiated in oocytes before development of patency in follicles of the moth, Plodia interpunctella. Roux’s Arch Dev Biol. 203:215–226.