Effects of precocene II on female-specific hemolymph polypeptides in Oncopeltus fasciatus.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 6:49-58 (1987) Effects of Precocene I I on Female-Specific Hemolymph Polypeptides in Oncopeltus fasciatus T. Martinez and M.D. Garcera Departamento de Quimica Orgdnim Biolbgica, Centro de Investigaci6n y Desarrollo (C. S.J. C.), Barcelona, and Departamento de Fisiologia Animal, Facultad de Ciencias Biol6gicas, Valencia, Spain Using polyacrylamide gel electrophoresis (PAGE) under denaturing conditions, two major polypeptides of 200,000 and 170,000 daltons were detected i n the hemolymph of mature female Oncopeltus fasciatus, but they were not found in the hemolymph of males or newly emerged females. Those polypeptides constituted the two major bands of early vitellogenic oocytes; however, they were absent from the yolk of mature eggs. The slower-migrating band (200,000 daltons) appears to correspond to a vitellogenic protein already identified in 0. fasciatus, whose synthesis has been suggested to be independent of juvenile hormone (JH).Treatment of newly emerged adult females with the corpus allatum cytotoxin precocene II prevented the appearance of the female-specific bands and induced an important accumulation of other proteins in the hemolymph. Yolk deposition was also inhibited in those animals. Topical application of JHto precocene-treated females restored the appearance of the 200,000 and 170,000 dalton polypeptides in the hemolymph. These results suggest that J His required for the synthesis of female-specific polypeptides in 0. fasciatus. Key words: juvenile hormone, vitellogenic oocytes, yolk proteins INTRODUCTION Juvenile hormone (JH) is necessary for egg maturation in most insects; however, its involvement in reproduction seems to vary from species to Acknowledgments: We thank Prof. W.S. Bowers for the generous gift of precocene [I, Dr. R. Martinez-Pardo for his interest in this work, and Dr. J.A. Veenstra for critically reading the manuscript. This study was supported by a grant from the Spanish Ministry of Education and Science to T.M. Received March 24,1986; accepted May 5,1987. Address reprint requests to T. Martinez, Dept. Qufmica Organica Biologica (C.S.I.C.), J. Cirona Salgado 18-26,08034 Barcelona, Spain. 0 1987 Alan R. Liss, Inc. 50 Martinez and Carcera species, and different control mechanisms of oogenesis have been proposed [l-31. In Heteroptera, evidence from experiments with allatectomized females treated with JH suggests that vitellogenin synthesis is regulated by JH in Dysdercus intermedius  and Triatomu protructu . Synthesis of vitellogenin could also be stimulated in males of Rhodnius proEixus with the JH analog methoprene . JH appears to be required, at least partially, for yolk deposition in the species of this order examined [5,7l. In Oncopeltus fusciatus, oviposition occurs at irregular intervals, but egg development seems to be a continuous process . Although this heteropteran species is also JH-dependent for vitellogenesis [8-lo], it is unusual in that an antigenically incomplete vitellogenin (vitellogenin A) appears in the hemolymph of diapausing females, before CA" activation [lo]. This protein is also present in precocious adults, resulting from treatment of larvae with precocene 11, animals in which the CA is supposed to be inactive [ll]. These observations suggested that synthesis of vitellogenin A could be independent of JH. Only after stimulation with JH did an antigenically complete vitellogenin (vitellogenin AB) appear in the hemolymph, probably resulting from cleavage of the A form. It is thought that it is the mature vitellogenin (AB), the protein incorporated by the oocytes with a further modification during uptake or oocyte maturation [l0,12]. In the present study, we electrophoretically analyze the effects of CA inactivation by precocene I1  on hemolymph and ovarian polypeptides of 0. fasciutus and present some evidence suggesting an influence of JH on the synthesis of major female-specific polypeptides, one of them putative vitellogenin A. MATERIALS AND METHODS Experimental Animals and Treatments Oncopeltus fusciatus was reared in the laboratory at 29°C _+ 1°C at 60-65% relative humidity, and 16 h photophase. Animals were fed on a mixed diet of milkweed and sunflower seeds, and water was supplied ad libitum. Precocene 11 Treatment Newly emerged adult females (0-2-h-old) were treated with precocene I1 (6,7-dimethoxy-2,2-dimethylchromene; a gift of W.S. Bowers) by the contact method . The animals were confined for 42 h in petri dishes pretreated with an acetone solution of 500 pg precocene I1 (7.86 pg/cm2).After treatment, females were transferred to normal rearing conditions, and males were added the following day. Control animals were maintained in petri dishes treated with acetone. JH Treatment To reverse the effects of precocene 11, JH I (R. Maag, Ltd., Dielsdorf, Switzerland) was topically applied to precocene-treated animals on the ab*Abbreviations: CA = corpus allatum; JH = juvenile hormone; PAGE = polyacrylamide gel electrophoresis; SDS = sodium dodecylsulfate. Precocene Effects on Female-Specific Polypeptides 51 dominal sternites. Fifteen females were treated immediately after precocene exposure; in 20 other females, JH application was initiated five days after precocene treatment. In both groups, 5 pg of JH I (in 0.5 pl acetone) was applied daily for four consecutive days. Control animals were treated with acetone after exposure to precocene 11. Sample Preparation Hemolymph (4 pl vol) was collected from the stubs of sectioned legs in glass capillary tubes coated with heparin, and it was diluted to a concentration of 10% with a solution containing 15 mh4 tricine, pH 7.4, 6.4% sucrose, 2 mM MgC1, and 20 mM KC1. Ovaries were dissected from adult females, washed several times in insect saline, and homogenized in 60 p1 of the same solution. Homogenates of different parts of the ovary were also prepared. Chorionated eggs from mature females were opened and yolk was collected. Samples were boiled for 4 min in 77 mM Tris HC1 pH 8.0, 1.8% SDS, 4.4% 2-mercaptoethanol, and 8.5% glycerol . They were stored at -20°C until use. Individual animals were used in the present study to electrophoreticdy analyze the protein content of hemolymph and ovary of the same female. Sample collection was performed at 5 day intervals, starting at day 5 until day 30 after precocene I1 treatment. Hemolymph and ovaries from five controls and from seven precocene-treated females were analyzed in every period (ie, 5, 10, 15, 20, 25, and 30 days after treatment). Collection of hemolymph in those females treated with JH I (or acetone) was performed five days after the initial dose (ie, 5 or 10 days after precocene treatment). SDS-PAGE Electrophoresis was performed under denaturing conditions at a constant current of 20 mA and 4"C, using the discontinuous system described by Conejero and Semancik . The resolving gel contained 9% acrylamide. Two microliters of 0.01% bromphenol blue was added to the samples before electrophoresis. Gels were fixed in an aqueous solution of 12.5% trichloroacetic acid and 25% isopropanol and stained with 0.05% Coomassie brilliant blue R 250. The molecular weight standards (Bio-Rad) used were: myosin (M, = 200,000), P-galactosidase (M, = 116,250), phosphorylase B (M, = 92,500), bovine serum albumin (M, = 66,200), and ovalbumin (M, = 45,000). RESULTS With the present electrophoretic conditions, we could clearly visualize in the hemolymph of mature 0. fasciatus females two major polypeptides with M,s of 200,000 and 170,000. These polypeptides were not detected in the hemolymph of adult males of aferent ages (Fig. 1).No appreciable concentrations were observed in the hemolymph either of fifth-instar nymphs or of newly emerged females, which showed a hemolymph electrophoretic pattern similar to that of males. The other characteristic polypeptides of mature female hemolymph appeared to be common to both sexes. Some of these bands were present in 52 Martinez and Carcera 5 l9 10 d"? 6 '3 20 15 d"? 25 6' ' 9 d' 200 1 , 170- Fig. 1. SDS-electrophoretic analysis of hemolymph of 0. fasciatus adult females and males. Four microliters of hemolymph in 55 pI volume (see text) were loaded per well. Number of days after the imaginal molt is indicated at top. Arrows show female-specific polypeptides with approximate molecular weights of 200,000 and 170,000 daltons. important concentrations in young mature females; however, they were greatly reduced in the hemolymph of females older than two weeks (eg, band 6 in Figs. 2 and 3). Other polypeptides appeared to be depleted from the hemolymph of females of that age (eg, bands 9 and 10 in Figs. 2 and 3). The electrophoresis of crude ovary extracts of reproductive females showed two major bands with the same mobilities as the female-specific hemolymph polypeptides (bands 2 and 3 in Fig. 2) suggesting that they result from the incorporation of the hemolymph polypeptides into the oocytes. However, the electrophoretic patterns of yolk from mature eggs or of whole eggs did not show those polypeptides (Fig. 4). Different regions of well developed ovaries were processed separately for electrophoresis in order to localize those bands within the ovary; we found that they constituted the major polypeptide bands of early vitellogenic oocytes (ca 0.5 mm in length). No appreciable concentrations of the yolk polypeptides of mature eggs were observed in those early follicles (Fig. 4). Effects of Precocene I1 Comparison of hemolymph electrophoretic patterns of control animals with those of females exposed to precocene I1 revealed important changes in the latter. The female-specific polypeptides were not detected in the hemolymph of any of the precocene-treated animals examined 5 or 10 days after exposure to precocene 11, whereas both polypeptide bands were always present in the controls (Fig. 2). The other characteristic hemolymph bands of young mature females were observed, however, in all experimental animals. Females examined from day 15 after precocene treatment revealed a conspicuous accumulation of nonspecific hemolymph polypeptides (Fig. 3). Some of these bands, which could hardly be detected in controls of the same age, were present in considerable concentrations in precocene-treated fe- Precocene Effects on Female-Specific Polypeptides 5 53 10 C PI I C PI I 1 2 3 10 U HF OV HF OV HF HF OV HF OV Fig. 2. SDS-PAGE of hemolymph (HF) and crude ovary extracts (OV) of 0. fasciatus adult females. Number of days after treatment i s indicated at top. The electrophoretic pattern of hemolymph with characteristic polypeptide bands (1-10) i s shown to the left. Arrows indicate the apparent absence of female-specific polypeptides (bands 2 and 3) from the hemolymph of precocene Il-treated animals (PII). C , acetone-treated animals. Note the lack of protein incorporation into the undeveloped ovaries of experimental females. males (eg, bands 9 and 10 in Fig. 3). Band 6, which typically appeared in low concentrations in the hemolymph of control females from the second period, largely accumulated in all precocene-treated animals examined (Fig, 3). It is worth noting that 8 of the 28 experimental animals examined later than two weeks after precocene treatment revealed the 17’0,000 dalton female-specific polypeptide in the hemolymph. Most of those females also showed the 200,000 dalton polypeptide (Fig. 3). Ovaries of precocene-treated animals were carefully examined under the microscope, and no maturing oocytes were found. The electrophoretic patterns of crude ovary extracts indirectly confirmed the CA inactivation by precocene 11, since no substantial protein incorporation was detected in those ovaries, even in the animals that showed the female-specific bands and a large protein accumulation in the hemolymph. JH Effects To confirm that the effects of precocene I1 on the female-specific hemolymph polypeptides were due to its allatotoxic action, we applied JH I to precocene-treated females. The hemolymph electrophoretic pattern of chemically allatectomized females posteriorly treated with JH revealed the presence of the two female-specific polypeptides both 5 and 10 days after precocene treatment. Control animals, treated with acetone after exposure to precocene 11, did not reveal, as before, any of those bands in the hemolymph (Fig. 5). 54 Martinez and Carcera 20 PI I OV C PI I HF HF OV HF OV Fig. 3. SDS-PACE of hemolymph (HF) and crude ovary extracts (OV) of 0. fasciatus adult females 20 days after treatment. Notice the accumulation of general polypeptides in the hemolymph of precocene-treated females (PII) and the presence of band 3 (a female-specific polypeptide) in one of them. C, acetone-treatedfemale. 2 200 3 4 .-( 4 116 93 66 45 Fig. 4. SDS-electrophoretic analysis of hemolymph and ovarian proteins of an 0. fasciatus mature female. 1: Yolk from mature eggs; 2: anterior region of ovarioles including vitellogenic oocytes (ca 0.5 mm); 3: vitellogenic oocytes (ca 0.5 mm); 4 hemolymph; S: molecular weight standards. Arrows indicate the female-specific polypeptides. Precocene Effects on Female-Specific Polypeptides 5 PI I 55 10 +JH +JH PI I Fig. 5. SDS-PACE of hemolymph of 0. fasciatus adult females treated with precocene II immediately after emergence. Number of days after precocene treatment is indicated at top. PII, precocene 11-treated animals; + JH, precocene Il-treated animals, later treated with JH I. Arrows indicate the two female-specific bands present in female hemolymph after JH application. DISCUSSION The major hemolymph polypeptides of 200,000 and 170,000 daltons from mature females constituted two main bands of crude ovary extracts. These observations suggested that those female-specific polypeptides could be vitellogenins (since they were both detected in the hernolymph and in the ovaries of reproductive females). Although they were not present in the yolk of mature eggs, they were found to be the main bands of early vitellogenic oocytes (ca 0.5 mm in length). These results indicate that the two femalespecific polypeptides are probably yolk precursors. The slower-migrating band possibly corresponds to a female-specific vitellogenic protein of 200,000 daltons identified in 0. fusciutus as vitellogenin A [10,12]. This protein, which is not detected in mature eggs, is thought to be a precursor form of an antigenically complete vitellogenin, called AB (or B), with a molecular weight of 68,000 daltons [10,12]. It has been suggested that vitellogenin A is cleaved in the hemolymph to produce vitellogenin AB and that this mature protein would be taken up by the oocytes in modified form, since the electrophoretic mobility and the peptide composition of the major yolk protein of mature eggs were found to be slightly different to those of vitellogenin AB [10,12]. The results obtained in the present study are in agreement with the above model in that the 200,000 dalton polypeptide, the putative vitellogenin A, may be a precursor of the mature yolk protein. However, results also indicate that the 200,000 dalton band could be incorporated directly into the oocytes and that the cleavage or molecular rearrangement might take place within the ovary during oocyte maturation. The 170,000 dalton female-specific polypeptide could be a vitellogenic protein as well and might correspond to a female-specific band that has been 56 Martinez and Garcera observed to migrate ahead of vitellogenin A . However, the lack of any further information about that protein precludes the possibility of establishing a relationship between these two bands. With the present electrophoretic conditions, we visualized a polypeptide band in female hemolymph with an approximate molecular weight of 68,000 daltons (band 6). However, a band with the same electrophoretic mobility was also found to be a major component of male hemolymph. No femalespecific polypeptide could be detected in that region of the gel, even after decreasing the concentration of acrylamide below 8%, as was suggested by Rankin and Jackle [El. We were unable, therefore, to confirm the presence of the mature form of vitellogenin (AB). The present results show some similarities with another heteropteran species, Dysdercus intemedius , in which the hemolymph electrophoretic patterns from males and vitellogenic females also differ in two major polypeptides (M, = 180,000 and 175,000).Those female-specific bands were found in yolk from both early vitellogenic follicles and mature eggs. The latter, however, also contained some polypeptide bands that were absent from female hemolymph, indicating a possible processing of vitellogenin during oocyte maturation in this species as well. Treatment of newly emerged adult females with precocene I1 inhibited the appearance of the 200,000 and 170,000 dalton female-specific polypeptides in the hemolymph of young mature females. JH application restored the appearance of both bands in the hemolymph. These results suggest at least a partial control by JH on the synthesis of major female-specific polypeptides in 0. fusciatus. It has been observed that vitellogenin synthesis in ethoxyprecocene-treated females of Locusta rnigrutoria could also be restored with the JH analog methoprene [lq. Previous studies by Kelly and Telfer [lo] revealed the presence of vitellogenin A in starved and diapausing females of Oncopeltus, animals with a supposedly inactive CA. These observations suggested that synthesis of vitellogenin A could be independent of JH. However, starved and diapausing females were found to have a certain JH titer in the hemolymph [18,19]. In addition, Rankin and Jackle  reported no evidence for JH control of vitellogenin synthesis in 0. fusciatus, since they could find both forms of vitellogenin in the hemolymph of females treated with precocene 11. However, in those experiments, precocene treatment was initiated 1-3 days after adult emergence, and it seems likely that this is too late to start treatment. We have observed that Oncopeltus females could be far along in ovarian development (ie, presence of advanced vitellogenic oocytes) when treatment with precocene I1 was initiated at that time of the adult age. This development could be due to the presence of some JH synthesized in those females after emergence, since vitellogenesis in Oncopeltus is known to be dependent on JH [8-lo]. In newly emerged 0. fusciutus females treated with precocene 11, degenerative changes of the CA parenchymal cells have been observed at the ultrastructural level; however, the extent of degeneration was found to vary in different cells 1201. It is possible then that some CA cells of precocene-treated females are producing JH. Precocene Effects on Female-Specific Polypeptides 57 Those previous reports showing the presence of vitellogenins in females with a CA thought to be inactive, together with our observations that the female-specific bands are present in the hemolymph of some precocenetreated animals examined some time (ie, 15-30 days) after treatment, indicate that low levels of JH may stimulate synthesis of vitellogenic proteins in 0. fusciutus. This possibility is also supported by the observation that vitellogenins appear in the hemolymph of 3-4-day-old females [ll], in which the JH titer is reported to be low . On the other hand, the important accumulation of other hemolymph polypeptides observed in precocene-treated females suggests that JH is not required, at least at high concentrations, for general protein synthesis in 0. fusciutus. A similar accumulation of proteins in hemolymph was reported in surgically allatectomized females of two orthopteran species [21,22]. However, major female-specific proteins, some of them identified as vitellogenins, were found to be absent from allatectomized females of different species [22-251. The lack of any important protein incorporation into the oocytes of all precocene-treated females examined is in agreement with previous findings in starved and diapausing females of 0.fusciutus, in which no yolk deposition was observed . These observations also indicate that in Oncopeltus, as in other insect species , the presence of proteins in the hemolymph cannot, by itself, induce yolk deposition. Although a certain JH titer is known to exist in starved and diapausing females [18,19], and probably in some precocene-treated animals, that titer appears not to be high enough to stimulate incorporation of proteins into the oocytes. The present results agree with the hypothesis that high levels of JH may be required for vitellogenin uptake in Oncopeltus, whereas low JH levels could be adequate for vitellogenin synthesis . 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