Immunological analysis of lipophorin in the haemolymph ovaries and testes of the fall webworm Hyphantria cunea (Drury).код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 27:153-I 67 (1 994) lmmunologicaI Analysis of Lipophorin in the Haemolymph, Ovaries, and Testes of the Fall Webworm, Hyphantria cunea (Drury) Hwa Kyung Yun, Woo Kap Kim, and H a k R. Kim Department of Biology, Korea University, Seoul, Korea Lipophorin (LP) was purified from haemolymph in last instar larvae of Hyphantria cunea (Drury) by KBr density gradient ultracentrifugation and gel filtration. LP is composed of Apo-LP I and Apo-LP II with molecular weights of 230 kDa and 80 kDa, respectively. The level of haemolymph LP in early pupae was somewhat greater than in last instar larvae. In males, this LP concentration is maintained throughout pupal development, whereas the level of haemolymph LP decreases in female pupae beginning at day 7, coincident with the onset of vitellogenesis in the fall webworm. In both male and female adults, haemolymph LP concentrations were dramatically increased in comparison to their pre-adult levels. Actually, LP was found in the ovary by irnmunodiffusion, tandem-crossed irnmunoelectrophoresis, and Western blotting. Location of LP in the ovary was also traced by immunogold labelling. Also, LP appeared in small amounts in protein yolk bodies of the ovary at an early stage of vitellogenesis, when nurse cells are bigger than the oocyte, but in greater amounts at those stages when the oocyte i s larger than nurse cells-that is, when vitellogenesis is actively taking place. This fact clearly reveals that LP i s synthesized by fat body and released into the haemolymph, and then taken up by the growing ovary during vitellogenesis. Also, LP was detected in testes by immunological analysis. Western blotting showed that LP was present in testicular fluid but not in the peritoneal sheath and cysts. To test whether LP i s also synthesized in testes, testes and fat body tissues were cultured in vitro, indicating that fat body synthesizes LP but testes do not. The result showed that the haemolymph LP itself i s taken up into the testes. o 1994 WiIey-Liss, Inc. Key words: haemolymph, fat body, testis, lipophorin, vitellogenesis Acknowledgments: The authors express their gratitude to Dr. Haruo Chino (Hokkaido University, Sapporo, Japan)for technical assistance in the purification of lipophorin and for careful review of the manuscript. This work was supported by the Basic Science Research Support Fund (1991)from the Ministry of Education, Korea. Received July 21, 1993; accepted December 6, 1993. Address reprint requests to Dr. Hak R. Kim, Department of Biology, Korea University, Seoul 136-701, Korea. 0 1994 Wiley-Liss, Inc. 154 Yun et a[. INTRODUCTION Lipophorin was shown to act as a vehicle for transporting lipid from fat body to flight muscle (Chino et al., 1977; Chino and Kitazawa, 1981; Van Heusden et al., 1987). Lipophorin consists of two different subunits called Apo-LP I and Apo-LP I1 (Chino et al., 1981; Shapiro et al., 1984; Robbs et al., 1985; Prasad et al., 1986; Fernando-Warnakulasuriya and Wells, 19881, and is combined with another subunit known as Apo-LP I11 or C protein when the insect is injected with adipokinetic hormone or during flight (Kawooya et al., 1984;Van der Horst et al., 1984;Wells et al., 1985; Haunerland et al., 1986). Lipophorin was also reported to shuttle lipid from fat body to ovary (Chino et al., 1981; Gondim et al., 1989) and was detected in the eggs of Philosamia silkworm (Chino et al., 1977).Further, high density lipophorin (HDLP)"of adult haemolymph was converted to very high density lipophorin (VHDLP) in the eggs of Manduca sexta (Kawooya et a]., 1988, Van Antwerpen and Law, 1992) and of Hyalophora cecropia (Telfer et al., 1991). However, detailed information on the involvement of lipophorin in vitellogenesis is still limited. Also, the presence and role of lipophorin in testes were not reported so far. The present work compares the concentration of lipophorin in haemolymph of males and females during vitellogenesis and proves uptake and location of lipophorin in growing oocytes of Hypha~triacunea (Drury). Also, the presence and uptake of Iipophorin in testes were immunologically examined. MATERIALS AND METHODS Insects Larvae of Hyphantria cunea (Drury) were reared on fresh willow leaves at 27 i 1°C and 75 5% relative humidity with a photoperiod of 16 h light and 8 h dark. Sexes were segregated during pupal stages. * Collection and Processing of Haemolymph, Ovary, Fat Body, and Testes Haemolymph was collected into cold test tubes by puncturing larvae and pupae with a needle. A few crystals of phenylthiourea were added to the tubes to prevent melanization. Haemolymph was centrifuged at 10,OOOg for 10 min at 4°C to remove haemocytes and cellular debris, and the supernatant was stored at -70°C until used. Ovary was dissected from 9-day-old pupae in cold Ringer's solution (128 mM NaC1,1.8 mM CaC12,1.3 mM KC1, pH 7.4). Some ovary samples were immediately used for electron microscopic observation. Others were homogenized and centrifuged at 10,OOOg for 10 min, and the supernatant was used in electrophoresis or immunological analysis. Fat body was dissected from larvae in cold Ringer's solution and homogenized. After centrifugation at 10,OOOg for 10 min, the supernatant was stored at -70°C until used. Testes were dissected from larvae and pupae in cold Ringer's solution and homogenized. After centrifugation at 10,OOOg for 10 min, the supernatant was *Abbreviations used: GAR = goat antirabbit; HDLP = high density lipophorin; HRP = horseradish peroxidase; LP = lipophorin; TBS = 20 mM tris, 500 mM NaCI, p H 7.5; TTBS = 0.05% Tween-20 in TBS; VHDLP = very high density lipophorin. Fall Webworrn Lipophorin 155 used in electrophoresis or immunological studies. For examination of different fractions, the testes were transferred to a measured volume of Grace’s medium (GIBCO, Grand Island, NY), and the sheaths were ruptured by teasing the organ apart with forceps, releasing the testicular fluid and cysts. These two components were resolved by a 2 min centrifugation at 200g, yielding a supernatant containing testicular fluid and a pellet consisting of cysts; the supernatant was transferred to a clean tube, and the cysts were washed twice by resuspension in Grace’s medium. The peritoneal sheath and cyst pellets were resuspended in a measured volume of cold Ringer’s solution and homogenized, and a soluble protein extract was collected as a post-12,OOOg supernatant. Purification of Lipophorin and Preparation of Antiserum Lipophorin was purified from the haemolymph of last instar larvae by KBr density gradient ultracentrifugation and Sephadex G-200 chromatography. Approximately 3 ml of the pooled haemolymph was centrifuged at 2,OOOg (5°C) for 5 min to remove the haemocytes, and 2.64 g of KBr was added with stirring to the supernatant to give a final density of 1.31 g/ml. The KBr-haemolymph mixture (6.5ml)was transferred to a 13ml centrifuge tube and overlayered with 6.5 ml of 0.9% NaCl (density 1.007 g/ml). The tube was placed in an ultracentrifuge TST41.14 rotor and centrifuged at 40,000 rpm for 16h at 4°C. The yellow lipophorin band was collected by pasteur pipette and passed through a Sephadex G-25 desalting column. Desalted lipophorin was eluted from a Sephadex G-200 (Pharmacia,LKB, Uppsala, Sweden) column (2 x 60 cm) with 0.05 M phosphate buffer (pH 7.0) at a flow rate of 30 ml/h, with 2 ml fractions collected. Purified lipophorin (400 pg/ml) was mixed with an equal volume of Freunds complete adjuvant and injected into a rabbit subcutaneously. Injections were given every other day for the first week, and a fourth injection was made 2 weeks later. Freunds incomplete adjuvant (0.5 ml) and purified lipophorin (400 pg/ml) were thoroughly mixed and used for a booster injection 2 weeks after the fourth injection. Blood was collected 1 week after the last injection and centrifuged at 10,OOOg for 10 min. The supernatant antiserum was stored at -70°C until used. Gel Electrophoresis and Western Blotting SDS-PAGE was conducted on a 10% SDS gel at room temperature at 15 mA according to the Laemmli procedure (1970). After electrophoresis, gels were stained in Coomassie brilliant blue R250. Molecular weight of the lipophorin subunits was determined as described by Lambin et al. (1976). Standard molecular weight marker proteins were used: myosin (Mr = 200 kDa), p-galactosidase (M, = 116 kDa), phosphorylase B (Mr = 97.4 kDa), bovine serum albumin (Mr = 66.2 kDa), and egg albumin (Mr = 45 kDa). After SDS-PAGE,some samples were transferred to a nitrocellulose sheet in Tris-glycine buffer (25 mM Tris, 92 mM glycine, 30% methanol, pH 8.3) at 100 V for 2 h (Towbin et al., 1979).After transfer, the nitrocellulose sheet was equilibrated in TBS for 10 min and incubated in blocking solution (3% gelatin in TBS) for 30 min. The sheet was then washed twice for 5 min with TTBS and incubated for 1h in a solution containing 300-fold diluted anti-lipophorin serum. The filter was again washed twice with TTBS and then incubated for 1 h in a solution containing 3,000-fold diluted 156 Yun et a!. Fraction Number Fig. 1. Gel-permeation chromatography of the LP fraction from KBr density gradient ultracentrifugation. The Sephadex C-200 column was eluted with 0.05 M phosphate buffer at the rate of 30 ml/h, and the eluates were collected in 2.0 ml fractions. Upper right panel: SDS-PAGE of Sephadex G-200 fractions. H: Last instar larval haemolymph. A: The peak fraction (A) from Sephadex (3-200. M: Standard molecular weight markers (myosin, 200 kDa; P-galactosidase, 11 6 kDa; phosphorylase B, 97.4 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 45 kDa). secondary antibody (GAR-HRP conjugated I&). After incubation and two more washes with TTBS, the sheet was submerged in HRP color development solution (60 mg color development reagent, 4-chloro-1-naphthol in 20 ml icecold methanol, plus 0.015% H 2 0 2 in 100 ml TBS). Development of purple color indicated binding of the primary antibodies. Immunological Analyses Immunodiffusion was conducted on 1%agarose containing 0.1% sodium azide (w/v) and veronal buffer (pH 8.6) as described by Ouchterlony (1949). The plates were stained in 1%amido black 1OB and destained in 2% acetic acid. Rocket immunoelectrophoresis was performed according to Laurel1 (1966). One percent agarose in 10 mM veronal buffer (pH 8.6) containing 0.1 % sodium azide was mixed with an appropriate amount of anti-lipophorin serum to yield Fall Webworm Lipophorin 157 Fig. 2. Rocket irnrnunoelectrophoresis analysis of haernolymph (4 pl) using anti-LP serum at different stages in male (1) and fernale (2) development. A: Last instar larvae. B: Prepupae. C: 2-day-old pupae. D: 4-day-old pupae. E: 6-day-old pupae. F: 8-day-old pupae. C:10-day-old pupae. H: 12-day-old pupae. I: Adult. 3% anti-lipophorin serum. Electrophoresis was conducted in 10 mM veronal buffer (pH 8.6) at 95 V for 4 h. Tandem-crossed immunoelectrophoresis was carried out according to the procedure of Axelson et al. (1973). Agarose, buffer solution, and antibody were the same as described for rocket immunoelectrophoresis. The first dimension was run at 10 V/cm for 3 h, and the second dimension was run at 5 V/cm for 18 h. 158 Yun etal. Electron Microscopic Observation by Immunogold Labelling Ovary and testes from pupae and flight muscle from adult were dissected in Ringer’s solution, and the tissues were pre-fixed in 2.5% glutaraldehyde for 2 h at 4°C. After washing with 0.1 M phosphate buffer (pH 7.2) three times at 15 min intervals, the tissues were dehydrated in an ethanol series. Dehydrated tissues were then placed in propylene oxide and embedded in an Epon-Araldite mixture. Embedded tissues were semithin-sectioned using an ultramicrotome (Sorvall MT-11), stained in 1%toluidine blue, and attached to thin section grids. These were washed with TBS, incubated in blocking solution for 20 min, and again washed with TBS. The grid was reacted with primary antibody solution diluted thirtyfold with antibody buffer and again washed with TBS three times to remove nonspecifically attached antibody. The grid was then reacted for 40 min with antirabbit IgG conjugated to protein-A gold particles (20 nm in diameter) (Sigma, St. Louis, MO) (Gosselin et al., 1984; Bendayan and Duhr, 1986).After washing with TBS and distilled water three times each, the grid was stained in 2% uranyl acetate and observed under a JEM 100 CX-I1 electron microscope (JEOL, Japan) at 80 kV. Controls included 1) use of secondary protein A-gold antiserum in the absence of treatment with primary antibody and 2) treatment of thin sections with colloidal gold alone. In Vitro Synthesis of Protein Fat body and testes were dissected in Ringer’s solution and preincubated in Grace’s insect medium (100 pl) for 10 min in a shaking incubator at 37°C. Afterwards, according to the protocol of Bownes (1982),[3%l-methionine(5 pCi) was added, and the tissues were incubated for an additional 4 h. After incubation, the samples were homogenized and centrifuged at l0,OOOg for 10 min. The supernatant was subjected to electrophoresisand autoradiography (Bonnerand Laskey, 1974). RESULTS Purification of Lipophorin Lipophorin was purified from the haemolymph of last instar larvae by KBr density gradient ultracentrifugation. The yellow band appeared at the upper half of the tube. LP is yellow in color due to the presence of carotenoids absorbed through food (Shapiro et al., 1984). This band was collected with a pasteur pipette and subjected to a Sephadex G-25 column to remove salts and then submitted to gel filtration (Sephadex G-200). Haemolymph and purified LP were applied to SDS-PAGE to confirm the purity of the LP fraction. Lipophorin Fig. 3. 1: lmmunodiffusion patterns with anti-LP serum. I: AB = anti-LP serum; A = purified LP; B = larval haemolymph; C = ovary extracts. II: AB = anti-LP serum; A = purified LP; B = larval haemolymph; C = pupal haernolymph in female; D = pupal haemolymph in male; E = adult haernolymph. 2: Tandem-crossed immunoelectrophoresis with anti-LP serum. A: Purified LP. B: Ovary extracts. C: Larval haemolymph. 3: SDS-PAGE (I) and Western blots (11) of purified LP (A), ovary extracts (B), and larval haemolyrnph (C). Standard molecular weight markers (M) are the same as those given in Figure 1. Fall Webworm Lipophorin 1 2 3 Figure 3 . 159 160 Yun et al. A B Figure 4. Fall Webworm Lipophorin 161 C Fig. 4. A:Thin section of adult flight muscle (I)and pupal ovary (11) treated with lipophorin antiserum and protein-A gold conjugate. The lipophorins were accumulated in protein bodies of ovary. P = protein body; L = lipid droplet; M =flight muscle. I:~16,000.II: ~17,500.B: Light micrographs of the ovaries of Hyphantria cunea at different stages. 1 : A longitudinal section of the stage 1 follicle. The oocyte is enclosed with the follicle cells. x400. 2: A longitudinal section of the stage 2 follicle. The length of the nurse cell is greater than that of the oocyte. x560. 3:A longitudinal section of the stage 3 follicle. The length of the oocyte is greater than that of the nurse cell. x700.4: A longitudinal section of the stage 4 follicle. The nurse cells are not observed. x770. NC = nurse cells; 0 = oocyte. C:Thin section of the ovaries of Hyphanfria cunea at different stages. The stages are the same as those given in 6. Original magnification ~5,300. P = protein body; L = lipid droplet; GP = glycogen particles. 162 Yun et al. was electrophoresed on 10% polyacrylamide gel to determine the number and molecular weights of its subunits. Lipophorin consists of Apo-LP I and Apo-LP 11, molecular weights of which were estimated to be 230 kDa and 80 kDa, respectively (Fig. 1). Concentration of Lipophorin in Haemolymph During Development Rocket immunoelectrophoresis was carried out to determine the relative concentration of LP in male and female haemolymph during developmental stages. Figure 2 shows that the level of haemolymph LP in early pupae was somewhat greater than in last instar larvae. In males, this LP concentration is maintained throughout pupal development (Fig. 2-11, whereas the levels of haemolymph LP decrease in female pupae beginning at day 7 (Fig. 2-2), coincident with the onset of vitellogenesis in the fall webworm. In both male and female adults, haemolymph LP concentrations were dramatically increased in comparison to their pre-adult levels. Ovarial Uptake and Location of Lipophorin Immunodiffusion, tandem-crossed immunoelectrophoresis, Western blotting, and immunogold labelling were performed to determine whether LP is present in the maturing ovary. Anti-LP serum made a continuous precipitine line with ovarial extracts, larval haemolymph, and male and female pupal haemolymph as well as purified LP (Fig. 3-1). Identity between LP and a component of ovarial extracts was also supported by the continuous arc between LP and ovarial extracts in the tandem-crossed electrophoresis experiment (Fig. 3-2). The presence of LP in ovary was further demonstrated by Western blotting (Fig. 3- 3). The location of ovarial LP was determined using protein-A gold particles linked to secondary (anti-rabbit Ig) antibodies. LP was accumulated in protein yolk bodies of the ovary but was not found in flight muscle (Fig. 4A). LP appeared in small amounts in protein yolk bodies of the ovary at an early stage of vitellogenesis, when nurse cells are bigger than the oocyte, but in greater amounts at those stages when the oocyte is larger than nurse cells (ie., actively undergoing yolk accumulation [Fig. 4B,Cl). Presence of Lipophorin in Testes LP was detected in testes by immunodiffusion, tandem-crossed immunoelectrophoresis, and Western blotting. Anti-LP antibodies yield a continuous precipitation arc between purified LP and testes extracts (Fig. 5A). The testes extracts were shown to contain Apo-LP I and Apo-LP I1 by Western blotting (Fig. 5B). Also, Western blotting was conducted to examine different fractions in testes. LP was detected in testicular fluid but not in peritoneal sheath and cysts (Fig. 6A,B). Fat body and testes dissected from last instar larvae were incubated in Grace’s medium containing [3%]-methionine.These tissues were homogenized in culture medium for electrophoresis and autoradiography. The lipophorin bands appeared in last instar larval fat body but not in larval and pupal testes (Fig. 7). This fact clearly reveals that LP is synthesized by fat body and released into the haemolymph, and then taken up by the testes. Fall Webworm Lipophorin 163 A B Fig. 5. A: Immunodiffusion (I) and tandem-crossed immunoelectrophoresis (11) with anti-LP serum. AB = anti-LP serum; A = purified LP; B = testes extracts. 6: SDS-PAGE (I) and Western blots (11) of purified LP (A) and testes extracts (8).Standard molecular weight markers (M) are the same as those given in Figure 1. DISCUSSION Generally, LP consists of Apo-LP I and Apo-LP 11, the molecular weights of which are 250 kDa and 78 kDa, respectively (Chinoand Kitazawa, 1981; Shapiro et al., 1984; Ryan et al., 1984).Lipophorin of H. cunea is also comprised of Apo-LP 1 and Apo-LP 11, with molecular weights estimated to be 230 kDa and 80 kDa, 164 Yun et al. A B Fig. 6. A: SDS-PAGE (I)and Western blots (11) of purified LP (A), peritoneal sheath (B), testicularfluid (C), and cysts (D). B: SDS-PAGE (I)and Western blots (11) of purified LP (L), testicular fluid from last instar larvae (A), 0-day-old pupae (B), 2-day-old pupae (C), 4-day-old pupae (D), 6-day-old pupae (E), and 8-day-old pupae (F). Standard molecular weight markers (M)are the same as those given in Figure 1. respectively. Although the concentration of LP during developmental stages has been little investigated, LP of Musca domestica showed a gradual increase but then a decrease during the adult stage (de Bianchi et al., 1987).However, the level of haemolymph LP in early pupae of H. mnea was somewhat greater than in last instar larvae. In males, this LP concentration is maintained throughout pupal development, whereas the levels of haemolymph LP decrease in female pupae beginning at day 7, coincident with the onset of vitellogenesis in the fall webworm. In both male and female adults, haemolymph LP concentrations were dramatically increased in comparison to their preadult levels (Fig. 2). Fall Webworm Lipophorin 165 Fig. 7. SDS-PAGE (I) and autoradiogram (11) of [35S]-methionine labelled proteins. A: Extracts of cultured last instar fat body. B: Extracts of cultured last instar larval whole testes. C: Extracts of cultured pupal whole testes. D: Purified LP. E,F,C: lmmunoprecipitated products of fat body, larval whole testes, and pupal whole testes, respectively. Standard molecular weight markers (M) are the same as those given in Figure 1. Lipophorin is well known to act as a vehicle transporting diacylglycerol from fat body to flight muscle (Chino and Kitazawa, 1981; Chino et al., 1981) and has been detected in the eggs of the Philosarniu silkworm (Chino et al., 1977). Also, HDLP of haemolymph is converted to VHDLP in the eggs of Manduca sexta (Kawooya et al., 1988). Thus, haemolymph LP was reported to be involved in yolk formation (Kawooya et al., 1988; Telfer et al., 1991;Van Antwerpen and Law, 1992). In the present work with H . cuneu, the presence of LP in oocytes was demonstrated by immunodiffusion, tandem-crossed immunoelectrophoresis, and Western blotting. Further, electron microscopic observation by immunogold labelling showed that LP was accumulated in protein yolk bodies of maturing oocytes but not found in flight muscle. This fact suggests that LP acts as a vehicle delivering lipid not to flight muscle but is totally uptaken into the ovary during vitellogenesis. Also, ovarial uptake of LP actively took place at the stage when the oocyte is bigger than nurse cells. This indicates that LP itself is actively involved in oocyte maturation during vitellogenesis. There have been no reports indicating that LP is present in testes. In the present work withH. cuneu, however, LP was detected in testes by immunological analysis. Also, LP was detected in testicular fluid in testes but not in the peritoneal sheath and cysts by Western blotting. Autoradiography was undertaken to test whether the LP present in testes comes from fat body via the haemolymph or from the testis itself. 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