Lipophorin-facilitated hydrocarbon uptake by oocytes in the German cockroach Blattella germanica (L.)
1 Department of Entomology, Box 7613, North Carolina State University, Raleigh, NC 27695-7613, USA and
2 Directorate for Education and Human Resources, National Science Foundation, 4201 Wilson Boulevard, Suite 875, Arlington, VA 22230, USA
* Present address: Paradigm Genetics, Research Triangle Park, NC 27709, USA
Author for correspondence (e-mail: coby_schal{at}ncsu.edu)
Accepted 21 December 2001
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Summary |
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Key words: Blattella germanica, cockroach, lipophorin, ovary, hydrocarbon transport, integument.
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Introduction |
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Studies in different groups of insects implicate the oenocytes, large abdominal cells rich in smooth endoplasmic reticulum and mitochondria, as the site of HC synthesis (Romer, 1991). The tissue localization of oenocytes varies considerably among insects, suggesting that the site of HC synthesis in insects varies with the location of oenocytes. In Tenebrio molitor, the oenocytes are grouped along the upper side of the tracheal trunks, separated from the fat body (Romer, 1991
), and in larvae of the butterfly Calpodes ethlius oenocytes are also found in the hemocoel (Jackson and Locke, 1989
). In the locusts Schistocerca gregaria and Locusta migratoria, the oenocytes are found only in the peripheral fat body, which is situated beneath the abdominal epidermis (Diehl, 1975
; Katase and Chino, 1984
). The oenocytes of Drosophila melanogaster are within the abdominal epidermis, and complete feminization of the HC pheromone mixture produced by males was recently induced by targeted expression of the transformer gene in adult oenocytes (Ferveur et al., 1997
), in support of the idea that oenocytes synthesize HCs. In the American and German cockroaches Periplaneta americana and Blattella germanica, respectively, the oenocytes are also distributed near the epidermal cells of the abdominal tergites and sternites and are separated from the hemocoel by a basal membrane (Kramer and Wigglesworth, 1950
; Liang and Schal, 1993
).
A large proportion of the HCs are directed to the exterior of the cuticle. Clearly then, when oenocytes are situated in the hemocoel, a hemolymph transport pathway would be required for delivery of HCs to the epidermis and ultimately to the epicuticle. Such a pathway, involving hemolymph high-density lipophorin (HDLp), has now been described in many insects (for a review, see Schal et al., 1998b). Interestingly, however, it is also prominent in insects whose oenocytes are adjacent to epidermal cells in the integument. In all studies that have examined internal lipids, a large but developmentally regulated pool of internal HCs has been found (Dwyer et al., 1986
; Guo and Blomquist, 1991
; Gu et al., 1995
; Young et al., 1999
; Sevala et al., 1999
, 2000
), and in most of these studies, the internal HCs appear to be qualitatively similar to the cuticular HCs (Chino and Downer, 1982
; Schal et al., 1994
, 1998b
; Gu et al., 1995
; Sevala et al., 2000
; Young et al., 2000
). The rationale proposed for internal HCs is that hemolymph HDLp functions primarily to deliver HCs from oenocytes to integumental tissues that do not synthesize HCs, including the head and thorax (including the legs and wings).
However, our studies of adult B. germanica females indicate that a major fraction of the internal HCs is localized within oocytes, suggesting that an important function of hemolymph HCs and HDLp is to provision the developing oocytes as well as cuticular surfaces overlying regions that do not synthesize HCs. Because neither the fat body nor the gonads can synthesize HCs, large amounts of HCs must be transported from the oenocytes and deposited in the ovaries prior to ovulation (Gu et al., 1995). Moreover, as large amounts of HCs are found in the egg case (Schal et al., 1994
, 1998b
; Young et al., 2000
), it appears that the female might provision her progeny with maternal HCs and possibly HDLp, the latter as a yolk protein precursor.
In many insects, almost half the yolk mass is composed of lipid imported by vitellogenin and lipophorin. Yet nothing is known about the mechanism by which HCs are deposited in insect oocytes, the role of HCs in embryonic development and HClipophorin interactions in the embryo. In this paper, we report the results of in vivo and in vitro analyses of HC transport and uptake by the ovary and the role of lipophorin in HC transport. We use the German cockroach B. germanica as a model system because (i) it is large and therefore readily amenable to physiological and biochemical approaches, (ii) its HCs have been chemically well characterized (Augustynowicz et al., 1987; Carlson and Brenner, 1988
; Jurenka et al., 1989
), (iii) the biochemical pathways of HC biosynthesis are well understood (Chase et al., 1990
, 1992
; Blomquist et al., 1993
), (iv) the sites and developmental time course of HC synthesis are better known than in any other arthropod (Gu et al., 1995
; Schal et al., 1994
; Young et al., 1999
), (v) HCs constitute the major lipid of lipophorin, representing up to 50 % of the lipids (Sevala et al., 1999
), (vi) the ovaries and integument take up massive amounts of HCs (Schal et al., 1998b
) and (vii) lipophorin is also taken up by the oocytes and therefore acts as a yolk protein precursor (see below). Thus, we combine the advantages of the well-characterized cockroach HCs and lipophorin system with physiological measurements and immunoassays to describe HC transport to the ovary.
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Materials and methods |
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Animals
The German cockroach Blattella germanica (L.) colony was maintained in incubators at 27±0.5°C, approximately 50 % relative humidity and on 12 h:12 h light:dark photoperiod. Newly emerged adult females were separated from the colony on the day of adult eclosion (day 0) and maintained in separate plastic cages. Insects were provided with Purina 5012 Rat Chow (Purina Mills, St Louis, MO, USA) and water ad libitum. Females were mated on day 6 and they oviposited 23 days later under these conditions. Females were always maintained in groups because solitary females are reproductively repressed (Gadot et al., 1989; Holbrook et al., 2000
).
Biosynthesis and transport of hydrocarbons in vivo and in vitro
Methylmalonyl CoA, derived from propionate, can serve as a methyl-branch donor in the synthesis of methyl-branched HCs in B. germanica (Chase et al., 1990). Since over 80 % of the HCs are methyl-branched (Jurenka et al., 1989
), overall HC synthesis can be followed. [1-14C]Propionate (16.65 kBq) in 0.5 µl of TC199 medium was injected into 5-day-old adult females. The females were provisioned with food and water in Petri dishes in an incubator at 27°C. Ovaries were dissected after 4 or 24 h, rinsed thoroughly at least three times in saline, and HCs were extracted from the ovaries and the rest of the body separately, purified and assayed for radioactivity. In vivo HC transport and uptake experiments were confirmed with the native HC 3,11-dimethylnonacosane. Synthetic racemic [11,12-3H]3,11-dimethylnonacosane (21.46 kBq) was either topically applied to the abdominal tergites or injected into the hemocoel in 0.4 µl of hexane. After predetermined incubation times, the ovaries were dissected, rinsed thoroughly, lipids were extracted, HCs purified and the radioactivity in the HC fraction analyzed by liquid scintillation spectrometry.
For in vitro studies, abdominal sternites 3 and 4 from 5-day-old virgin females were co-incubated with a pair of ovaries of various ages in 500 µl of TC199 medium [adjusted to 410 mosmol l1 by the addition of 55 mmol l1 NaCl and 40 mmol l1 Hepes, pH 7.4, and sterilized by filtration through a 0.22 µm low protein binding filter (Millipore, Bedford, MA, USA) just prior to use] and 25.9 kBq of [1-14C]propionate. All incubations were at 27°C with constant shaking on an orbital waving shaker (VWR, Atlanta, GA, USA) to oxygenate the tissues. After 4 or 24 h incubations, the tissues were removed and analyzed for labeled HCs. As required, either purified HDLp or hemolymph was added to the medium. These experiments were repeated with labeled HC. Sternites 3 and 4 from 5-day-old virgin females were dissected and rinsed thoroughly in saline, and [3H]3,11-dimethylnonacosane (21.46 kBq) was topically applied in 0.5 µl of hexane on the cuticular surface of the sternites. The sternites were co-incubated with ovaries in TC199 and processed as above.
Extraction and quantification of hydrocarbons
The surface lipids were extracted from egg cases as described by Young and Schal (1997). Briefly, each egg case was immersed in 2 ml of n-hexane containing 15 µg of n-hexacosane as internal standard, agitated gently for 5 min, and the solvent was decanted into a clean vial. This procedure was repeated, and the egg case was subjected to a final rinse with 1 ml of hexane. The three hexane extracts were combined and subjected to HC purification and analysis by gas chromatography.
Internal lipids were extracted by a modified Bligh and Dyer (1959) procedure (Gu et al., 1995
). For samples to be analyzed by gas chromatography, 30 µg of n-hexacosane was added as internal standard. Lipids were extracted from various tissues by homogenization in water for 30 s (Kontes micro ultrasonic cell disruptor, Vineland, NJ, USA), and the homogenate was extracted with hexane:methanol:water (2:1:1). Samples were vortexed vigorously and centrifuged at 2000 g (IEC-Centra7, International Equipment, Needham Heights, MA, USA) for 10 min. A sample of the hexane phase was loaded onto a Biosil-A (Bio-Rad, Richmond, CA, USA) mini-column (approximately 500 mg of silica gel in a glass-wool-stoppered Pasteur pipette), and the HCs were eluted with 7 ml of hexane. The solvent was reduced with a gentle stream of N2, and unlabeled HCs were quantified by gas chromatography whereas radioactive HCs were assayed in 3 ml of scintillation fluid (ScintiSafe EconoF, Fisher) by liquid scintillation spectrometry (LS5801, Beckman, Fullerton, PA, USA).
For gas chromatography analysis, the hexane was reduced to 12 µl with N2 and analyzed on an HP 5890II (Hewlett-Packard, Palo Alto, CA, USA) gas chromatograph equipped with a flame-ionization detector and interfaced with an HP 3365II ChemStation. Splitless injection was made into a 25 mx0.32 mmx1 µm HP-1 capillary column operated at 150°C for 2 min, then increased at 10°C min1 to 280°C and held for 10 min. The injector and detector were held at 280 and 300°C, respectively.
Hemolymph collection and lipophorin isolation and purification
Hemolymph was collected from ice-anesthetized day 24 females by severing the cerci and gently pressing the abdomen. Hemolymph was collected into chilled 1.5 ml microcentrifuge tubes containing cold saline and protease inhibitors (0.05 mol l1 sodium phosphate buffer, pH 7.0, containing 0.15 mol l1 NaCl, 10 mmol l1 EDTA, 5 mmol l1 glutathione, 2 mmol l1 phenylmethyl sulfonyl fluoride, 10 µg ml1 leupeptin and 10 µg ml1 pepstatin), centrifuged at 735 g for 2 min at 4°C to pellet the hemocytes, and the plasma was stored at 80°C.
HDLp was purified by KBr density-gradient ultracentrifugation, as described by Shapiro et al. (1984) and previously applied to B. germanica by Gu et al. (1995
) and Sevala et al. (1997
, 1999
). Briefly, plasma obtained from virgin females was mixed with 2.58 g of KBr in saline and adjusted to 5.8 ml. The KBr mixture was placed into a Beckman 13.5 ml QuickSeal tube, overlaid with 7.7 ml of freshly prepared 0.9 % NaCl, and subjected to ultracentrifugation with slow acceleration and deceleration at 285 000 g for 22 h at 4°C in a Beckman L8-70M ultracentrifuge using a fixed angle rotor (70.1 Ti). Fractions of 400 µl were collected starting from the top of the tube. The purity of lipophorin fractions was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). Pure lipophorin fractions were pooled, concentrated and dialyzed against 10 mmol l1 phosphate-buffered saline, pH 7.4, using a Centricon-10 microconcentration tube with a 10x103 molecular mass cut-off membrane (Amicon, Danvers, MA, USA). Protein concentration was measured by the Bradford method (Bradford, 1976
) with bovine serum albumin as standard.
Enzyme-linked immunosorbent assay (ELISA)
The lipophorin titer in hemolymph, ovaries and egg cases was determined by indirect ELISA as described by Sevala et al. (1999), with minor modifications. Diluted (1:2000 to 1:8000) hemolymph (100 µl), ovary extract or egg case extract and a series of HDLp standards (1100 ng ml1) in coating buffer (50 mmol l1 sodium carbonate/bicarbonate buffer, pH 9.4) were bound to each well of an Immunoware high-binding 96-well ELISA plate (Pierce, Rockford, IL, USA) by incubating overnight at 4°C. Several blanks were also included in each plate. The plates were rinsed three times with phosphate-buffered saline containing Tween-20 (8 mmol l1 sodium phosphate, 2 mmol l1 potassium phosphate, 140 mmol l1 sodium chloride, 10 mmol l1 potassium chloride and 0.05 % Tween-20, pH 7.4) and blocked for 1 h with 1 % bovine serum albumin at 37°C. The wells were then filled with 100 µl of diluted HDLp antiserum in phosphate-buffered saline containing Tween-20 (1:500) and normal rabbit serum, and incubated for 1 h. Plates were washed three times and loaded with 100 µl of anti-rabbit immunoglobulin conjugated to alkaline phosphatase (Sigma) diluted 1:10 000 in phosphate-buffered saline containing Tween-20, and incubated for 1 h at 37°C. Plates were washed again and developed at room temperature with 100 µl of enzyme substrate, p-nitrophenyl phosphate (Pierce), and the reaction was stopped after 30 min by adding 100 µl of 2 mol l1 NaOH to each well. Absorbance was read at 405 nm in a PowerWave200 automated microplate reader (Bio-Tek, Winooski, VT).
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Results |
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These data, coupled with previous results showing that the ovaries do not synthesize HCs (Gu et al., 1995), demonstrate that HCs must be shuttled to the ovaries through the hemolymph. Moreover, transfer of ovarian HCs to the interior of the egg case suggests that ovarian HCs are deposited into the maturing basal oocytes and not the follicle cells that encase them. We examined the hemolymphovary transport pathway both in vivo and in vitro.
In vivo biosynthesis and transport of hydrocarbons to the ovaries
Because HC synthesis is developmentally regulated in adult females (Schal et al., 1994), females of different ages were injected with 21.46 kBq of [3H]3,11-dimethylnonacosane, the major HC in the native HC profile, to control for HC synthesis and therefore examine only its distribution within the female. Females were incubated for 24 h at 27°C, their epicuticular lipids extracted with hexane, the ovaries were dissected and thoroughly rinsed, and cuticular and ovarian HCs were purified and assayed by liquid scintillation spectrometry. HC uptake by the ovaries was low on days 02, increased on days 34, peaked on days 56 and declined sharply thereafter (Fig. 2A). The ovaries of pregnant females took up no HCs, as was also indicated by the gas chromatography data (Fig. 1), and neither did the egg case on days 16 and 26; the exterior of these egg cases contained approximately 200 disints min1 of HC (data not shown), probably from contamination from the cuticular surface.
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We compared the time course of HC biosynthesis and its deposition in the ovaries by measuring the incorporation of [14C]propionate into HCs in the ovaries and the rest of the body 4 and 24 h after injection. Young and Schal (1997) showed that, after injection of radiolabeled propionate into B. germanica nymphs, incorporation into HCs was complete within 24 h. The in vivo data presented in Table 1 confirm that HC biosynthesis was rapid. Within 4 h, 132x103 disints min1 was incorporated into HCs and no further HCs were synthesized by 24 h (116 978 disints min1; P>0.05; Students t-test). However, the distribution of the newly biosynthesized methyl-branched HCs changed during the 24 h incubation. While the amount of labeled HCs in the ovaries increased 3.7-fold, the amount of labeled HCs in the rest of the body declined by 23.3 % at 24 h (Table 1).
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Involvement of lipophorin in hydrocarbon transport to the ovaries
The hydrophobic nature of HCs suggests that plasma lipoproteins might shuttle them from the integument to storage and deposition sites. Indeed, in a number of insect species, hemolymph HCs associate with an HDLp (Chino, 1985; Schal et al., 1998b
; Sevala et al., 2000
). We tested the hypothesis that HDLp delivers HCs to oocytes by quantifying HC uptake by the ovary in the presence of either hemolymph or HDLp. Sternites 3 and 4 from 4-day-old virgin females were co-incubated for 24 h with an ovary from day 5 females in the presence of 25.9 kBq of [1-14C]propionate. With TC199 medium alone, large amounts of HCs were biosynthesized (95 918±24 946 disints min1, mean ± S.E.M., N=5), but most of the HCs remained with the sternites and only 78±29 disints min1 was deposited into the ovary; the amount deposited in the ovary was subtracted as background from all other incubations. The amount of labeled HCs in the ovary increased with increasing hemolymph concentrations in TC199, reaching a plateau at 10 % hemolymph (Fig. 3).
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The relationship between ovarian uptake of HCs and ovarian age was examined in vitro using co-incubations of sternites 3 and 4 from day 5 virgin females together with ovaries of various ages and 1 mg ml1 HDLp. [3H]3,11-Dimethylnonacosane was topically loaded onto the sternites so that only transfer of labeled HC to the ovary was considered. As expected from the in vivo results (Figs 1, 2), HC uptake increased steadily with growth of the ovary and peaked on day 8, just before ovulation; it declined sharply after oviposition on day 9 (Fig. 5A). To relate ovarian uptake of HCs to their physiological stage, ovaries from day 69 females were co-incubated in vitro with sternites from day 5 females, [14C]propionate and 10 % hemolymph. Day 7 ovaries took up the largest amount of 14C-labeled HCs (Fig. 5B).
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Discussion |
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Our previous investigations with B. germanica revealed more than 400 µg of internal HCs just before ovulation (Schal et al., 1994). This amount declined dramatically after oviposition, suggesting that the eggs were provisioned with HCs and constituted a major fraction of internal HCs in adult females. Herein, we confirmed that 232 µg of these internal HCs is deposited in the ovaries, much more than the less than 160 µg in the hemolymph (Fig. 1) or approximately 150 µg found on the epicuticular surface of the female (Schal et al., 1994
; Young et al., 1999
). Moreover, we showed that all ovarian HCs were transferred to the egg case, confirming that HCs are localized within the oocytes and not in other ovarian tissues, such as follicle cells. Since the ovaries do not synthesize HCs de novo (Gu et al., 1995
), a hemolymph transport pathway must be involved, and our results demonstrate that hemolymph is indeed required to effect the transfer of HCs from the sternal integument (probably oenocytes) to the ovary (Fig. 3).
Seminal work by Chino and co-workers showed that in several insect species hemolymph HCs are associated with HDLp, a major hemolymph lipoprotein of 600 kDa. In most insects, HDLp is characterized by two constituent apoproteins, apoLp-I and apoLp-II, with approximate molecular masses of 240 kDa and 80 kDa, respectively (Fig. 4A) (Chino et al., 1981; Chino, 1985
; Law and Wells, 1989
; Kanost et al., 1990
; Law et al., 1992
; Van der Horst et al., 1993
; Blacklock and Ryan, 1994
; Soulages and Wells, 1994
; Arrese et al., 2000
). HDLp serves multiple functions, including that of a juvenile hormone binding protein in beetles, termites, flies, bees and cockroaches (Trowell, 1992
; King and Tobe, 1993
; Sevala et al., 1997
; Engelmann and Mala, 2000
). Its most recognized function, however, is in bulk transport of lipids, including phospholipids, diacylglycerol (DAG), carotenoids, hydrocarbons and, in some mosquitoes, triacylglycerol (Ford and Van Heusden, 1994
). Lipids may comprise up to 50 % of the mass of lipophorin, but its lipid composition varies greatly among species and probably in relation to physiological stages.
In many insects, HCs constitute the major lipid carried by HDLp, and the dynamic transformations of lipophorin may be more closely related to reproductive physiology in adults and the periodic replenishment of HCs in the new cuticle of immatures than with DAG-fueled flight. In cockroaches, termites, locusts, beetles, flies and moths, HDLp shuttles HCs to the cuticle, and in some insects, to specialized pheromone glands (for reviews, see Schal et al., 1998a,b
). The fat body, too, contains HCs (Gu et al., 1995
; Young et al., 1999
), and our present results show that the ovaries also serve as an important HC deposition site in females. Because lipophorin is the major HC transporter in the hemolymph, the HC profiles of the hemolymph, lipophorin and cuticle are generally similar (e.g. Chino and Downer, 1982
; Schal et al., 1998b
; Sevala et al., 2000
). In some insects, male and female HDLp carry different HCs, some of which serve as sex pheromones, and the sex-specific composition of lipophorin is determined by the types of HCs biosynthesized by sex-specific oenocytes [for example, cockroach and locust (Katase and Chino, 1984
; Gu et al., 1995
), Drosophila melanogaster (Pho et al., 1996
) and moths (Schal et al., 1998a
)] and not by features of lipophorin.
In the moth M. sexta, a lipid transfer particle (LTP) plays a role in the delivery of lipids to the developing oocytes and the conversion of adult HDLp to egg very high density lipophorin (Liu and Ryan, 1991). In some insects, LTP also carries HCs (Blacklock and Ryan, 1994
), and in the American cockroach HCs comprise 40 % of the lipids in LTP, while HDLp carries only 28 % of HCs; however, the latter is much more abundant in the hemolymph (Takeuchi and Chino, 1993
). However, our results, together with previous in vitro work with purified HDLp (Katase and Chino, 1982
, 1984
), suggest that LTP is not necessary for exchange of HCs between the integument and HDLp and between HDLp and the ovary. We found maximum HC uptake by the ovary from sternites with either 10 % hemolymph (Fig. 3) or 1 mg ml1 HDLp (Fig. 4C). Since the concentration of HDLp in the hemolymph of the German cockroach is approximately 9 mg ml1 (Sevala et al., 1999
), the in vitro uptake data agree well with the in situ conditions. Nevertheless, it is possible that LTP may be associated with, or even synthesized by, the incubated tissues, and these experiments will have to be repeated with LTP antibodies to block LTP function.
Unlike vertebrate lipoproteins, insect lipophorin is generally considered to be a reusable particle that shuttles lipids among tissues without entering cells (Chino and Kitazawa, 1981; Van Heusden et al., 1991
). Chino et al. (1977
) suggested that lipophorin must act as a reusable lipid shuttle because insect eggs contain larger amounts of lipid than could be accounted for by ovarian lipophorin and vitellogenin. Indeed, in some insects, no lipophorin can be detected in oocytes or it represents only a minor fraction of total egg protein, for example in Rhodnius prolixus (Gondim et al., 1989
), suggesting that lipophorin acts primarily as a shuttle. In other insects, however, oocytes sequester lipophorin, and ovarian lipophorin is physically and immunologically identical to hemolymph lipophorin, suggesting a common origin (Thomas and Gilbert, 1969
; Chino et al., 1977
). Furthermore, immunocytological observations and tracking of [3H]DAG-labeled lipophorin and [35S]apoLp-labeled lipophorin showed that HDLp from adult M. sexta hemolymph was sequestered by oocytes without recycling the lipophorin back to the hemolymph (Kawooya and Law, 1988
; Kawooya et al., 1988
; Van Antwerpen et al., 1993
). In the moth Hyalophora cecropia, HDLp and vitellogenin enter the oocytes through the same endocytic mechanism, presumably involving receptor-mediated processes; HDLp is then converted into very high density particles within the eggs (Kulakosky and Telfer, 1990
; Telfer and Pan, 1988
; Telfer et al., 1991
). The clearest documentation of lipophorin uptake into oocytes comes from recent studies of the mosquito Aedes aegypti. During the first vitellogenic cycle, Lp gene expression is upregulated after a blood meal and hemolymph lipophorin is transported into the yolk granules of the developing oocyte (Sun et al., 2000
). The lipophorin receptor in the mosquito oocyte is distinct from the vitellogenin receptor (Cheon et al., 2001
), unlike in Hyalophora cecropia oocytes (Kulakosky and Telfer, 1990
).
An early study identified non-vitellogenin proteins with a high lipid content, presumably lipophorin, in ovaries of B. germanica (Kunkel and Pan, 1976). Our polyclonal antibodies against hemolymph HDLp recognize ovarian proteins in this species (Fig. 6), suggesting that lipophorin enters the oocytes. Yet, the amount of lipophorin in the ovaries appears to be low relative to the amount of vitellin, and a calculation of the HC:lipophorin ratio suggests that not all HCs enter the oocytes with internalized lipophorin. We recovered 232 µg of HCs from a mature ovary pair containing approximately 40 basal oocytes (Fig. 1), but only 65 µg of lipophorin (Fig. 6). Since all hemolymph HCs are carried by HDLp, and the hemolymph contains approximately 9 µg HDLp µl1 and approximately 10 µg HCs µl1 (Sevala et al., 1999
), it follows that 1 µg of HDLp carries approximately 1 µg of HCs. Clearly, HDLp must either unload the bulk of the HCs at the oocyte surface (as a reusable shuttle) or enter the basal oocytes, unload HCs and other lipids and recycle to the hemolymph. Regardless, the age-related uptake of HC and HDLp in the ovaries is identical to vitellogenin uptake, suggesting either that common receptors are used or a coordinated proliferation of independent receptors for vitellogenin and lipophorin. Interestingly, it appears that, in Manduca sexta, 90 % of the lipids that accumulate in the egg are delivered by low-density lipophorin that is not internalized (Kawooya and Law, 1988
). Cockroaches do not have low-density lipophorin, leaving open the question of how HCs might be deposited in the ovaries.
In the cockroach Diploptera punctata, HDLp content increases to approximately 1.5 µg per oocyte early in vitellogenesis, but declines rapidly to non-detectable levels in ovulated eggs (King and Tobe, 1993). This pattern indicates that lipophorin shuttles out of the oocytes, that it is rapidly metabolized within the oocytes or that lipophorin is associated only with follicle cells and not with oocytes. Notably, in this viviparous cockroach, the bulk of maternal lipids is taken up by embryos, not oocytes. In ovoviviparous Leucophaea maderae, much smaller amounts of lipophorin than vitellogenin enter the eggs, as in B. germanica, but nothing is known about its ovarian HCs. It would be of interest to relate the reproductive mode of cockroaches (oviparity, ovoviviparity, viviparity) to maternal strategies of provisioning HCs into the eggs. The relatively high HC content of B. germanica HDLp, compared with other insects, including other cockroaches (e.g. Periplaneta americana) (Chino and Kitazawa, 1981
), is probably related to its role in supplying HCs to a large batch of externally incubated oocytes, and it is possible that less HCs are provisioned in cockroaches that internally incubate their embryos.
It follows then, that in B. germanica, even though HDLp serves many functions, including that of a juvenile-hormone-binding protein, its titer will fluctuate in relation to shuttling HCs (Sevala et al., 1999). Soon after eclosion, the teneral cuticle has a large demand for HCs (Fig. 2B) (Schal et al., 1994
). As the oocytes begin to grow, they too sequester large amounts of HCs, and the female meets these increasing demands for apolar lipids by producing more HCs and HDLp, elevating hemolymph volume (Sevala et al., 1999
) and redirecting HC deposition away from the cuticle (Fig. 2B).
The mechanism(s) of HDLp-mediated delivery of HCs to oocytes, the function(s) of lipophorin within the oocyte and the fate of ovarian lipophorin and HCs have not been studied in any insect, and recent investigations of lipid accumulation in the ovaries of other arthropods have yet to identify lipoprotein carriers (Ravid et al., 1999). It is reasonable to hypothesize that maternal HCs serve to provision the embryonic and neonate cuticle with a waterproofing layer. We have shown previously that the female provisions the exterior of her egg case with an HC profile that has a melting temperature 15°C higher than that of her own cuticular HCs; this would provide the egg case with superior waterproofing (Young et al., 2000
). Our preliminary radiotracer experiments show that maternal HCs that are deposited in the egg appear on the neonate epicuticle (C. Schal, unpublished results). Moreover, although the embryo can synthesize HCs de novo around dorsal closure (Y. Fan and C. Schal, unpublished results), their contribution to the neonate cuticle is relatively minor. Our working hypothesis, thus, is that maternal lipophorin within the embryo serves to transport maternally sequestered HCs to the embryonic and neonate cuticles.
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Acknowledgments |
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References |
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