GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

85,286-296 (1992)

Role of the Midgut Gland in Metabolism and Excretion Ecdysteroids by Lobsters, Homarus americanus MARK Bodega

Marine

Laboratory,

J. SNYDER’ Universify

AND ERNEST

of California,

P.O.

of

S. CHANG’

Box 247, Bodega

Bay,

California

9492.3

Accepted February 27, 1991 The chromatographic profile of ecdysteroids (Ecds) from the midgut gland (MC) of juvenile female lobsters, Homarus americanus, was examined using high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA) over four stages of the molt cycle. Upon initial examination, highly polar Ecd conjugates appeared to be the principal metabolites found in all molt stages. HPLC fractions containing apolar Ecds initially exhibited low RIA activity. Upon hydrolysis with a He/ix pomatia enzyme preparation and reanalysis, significant amounts of other Ecds were released. Amounts of apolar Ecd conjugates were estimated, at their highest levels, to be at least 50% of the total Ecds in MGs of molt stage D, lobsters. Only the MG formed significant amounts of apolar Ecds upon in vitro culture with [3H]ecdysone ([3H]E). Epidermis and antenna1 gland significantly increased their rates of t3H]E metabolism in vitro between molt stages C, and D,. This result further supports the idea that regulation of ecdysteroid metabolism, at least in selected tissues, may be important in the molt cycle regulation of hormone titers. Using gel filtration column chromatography and sucrose density gradient centrifugation analyses, evidence was found for association of apolar Ecds with a protein(s) from MC cytosol. The protein was estimated to have a molecular weight of 180,000-200,000 and specifically bound apolar Ecds. o IWZ Academic

Ecdysteroids (Ecds) are polyhydroxylated steroid hormones found in arthropods. In crustaceans, Ecds are known to be important in the regulation of molting and may have other roles in reproduction (see Chang, 1989; Lachaise, 1989, for recent reviews). Ecdysone (E) is generally considered the primary product secreted by the crustacean Y-organ (Chang and O’Connor, 1977). Recent evidence suggests that several other Ecds, namely, 25deoxyecdysone and 3-dehydroecdysone, may also be produced by Y-organs (Lachaise et al., 1989; Spaziani et al., 1989). The initial studies of arthropod Ecd metabolism, using [3H]E, revealed that the pri-

mary metabolite, 20-hydroxyecdysone (20E), was produced by a single hydroxylation step. Subsequent metabolites appeared to be of greater polarity than 20E (King and Siddall, 1969; Moriyama et al., 1970). Several short-term in vivo labeling studies on decapod crustaceans demonstrated equivalent types of Ecd metabolite profiles (Lachaise et al., 1976; Kuppert et al., 1978; Buchholz, 1982). McCarthy (1980, 1982) and Lachaise and Lafont (1984) identified other products of E3H]E, L3H]20E, or [3H]ponasterone A ([3H]P) as 20,26-dihydroxyecdysone and various highly polar conjugates. Recently, we (Snyder and Chang, 1991a,b,c) showed that these products were present in hemolymph, urine, and feces of the lobster, Homarus americanus, and demonstrated a route in the lobster gut that removed Ecds from either hemolymph or ingested sources and

’ Present address: Department of Entomology, University of Arizona, Tucson, AZ 85721. ’ To whom all correspondence should be addressed. 286 0016~6480/92 $1.50 Copyright 0 1992 by Academic Press, Inc. Au rights of reproduction in any form reserved.

MIDGUT

GLAND

converted them to apolar conjugates prior to excretion in the feces. This apolar pathway of Ecd metabolism has been found in whole body extracts, ovaries, deposited eggs, and feces of a variety of noncrustacean arthropods (Isaac and Slinger, 1989). Besides identification and rates of metabolite formation studies, investigators have concentrated on the hemolymph transport and cellular dynamics of Ecds (Chang and O’Connor, 1988). Several laboratories have failed to demonstrate a hemolymph binding protein for Ecds in crustaceans (Chang et al., 1976; Kuppert et al., 1978; Lachaise, 1981). The regulation of Ecd activity at the cellular level may involve the cell membrane. Daig and Spindler (1983) reported evidence for carrier-mediated, energydependent uptake of Ecds by the decapod crustacean epidermis. In addition to membrane effects, cellular Ecd activities involve specific receptors, from both nuclear and cytosolic extracts, that have been found in several species including crayfish, crab, and brine shrimp (Spindler et al., 1984). Only data on binding affinities and approximate sizes obtained by velocity sedimentation centrifugation have been reported for crustacean cellular Ecd binding proteins (see Chang and O’Connor, 1988, for review). The present study extends earlier observations on the distribution and metabolism of Ecds in lobsters (Snyder and Chang, 1991a,b,c). Based on earlier evidence that the midgut gland (MG) was a major tissue for both Ecd uptake and metabolism (Snyder and Chang, 1991c), we now report titers of Ecd metabolites in midgut glands (MGs) of lobsters at four molt stages. Rates of in vitro [3H]E metabolism by selected lobster tissues at different molt stages were also determined. In addition, we report evidence for apolar Ecd association with a putative binding protein in the lobster that is similar to one previously described in the crayfish (Gore11 et al., 1972).

287

ECDYSTEROIDS

MATERIALS

AND METHODS

Animals. Juvenile female lobsters, Homarus icanus (35-120 g wet wt) were reared at the

amer-

Bodega Marine Laboratory using established techniques (Chang and Conklin, 1983; Conklin and Chang, 1983). They were maintained under previously described conditions (Chang and Bruce, 1980). Molt staging was conducted using previously published methods (Aiken, 1973). The early premolt stage D, is equivalent to the lobster stages D,’ to D,” of Aiken (1973). Stage D, refers to the premolt stages D,‘” of Cheng and Chang (1991). Postmolt stage A was assigned to lobsters that completed ecdysis within 24 hr prior to sampling. Tissue incubations. Abdominal muscle, MG, hindgut, and antenna1 glands were removed from juvenile lobsters (in molt stages A, C, D,, or D3), rinsed with lobster saline (Mykles, 1980) and cultured in saline containing 0.1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma). [3H]Ecdysone (New England Nuclear, high-performance liquid chromatography purified) was dissolved in lobster saline, added to the cultures, and incubated at 20” for 12 hr. Tissues were homogenized in methanol by 20 strokes using a Dounce homogenizer. Culture media and tissue homogenates were extracted twice with methanol followed by centrifugation at 4100g for 15 min. High-performance

liquid

chromatography

(HPLC).

Homogenates of incubated tissues or unincubated MGs were extracted in methanol and centrifuged, and the supematants were injected directly onto a Waters C,, PBondapak column (3.9 mm i.d. x 30 cm). Reverse phase (RP) elutions were conducted by one of the following: (1) a 35min linear gradient of 20-100% acetonitrile in 20 mM Tris, pH 7.5, at 1.Oml/min, or (2) a 35-min linear gradient of 20-100% methanol in 20 mM Tris, pH 7.5, at 1.0 mlimin (either 0.5- or l.O-min fractions for each system), both using a Waters HPLC system. Tentative identification of Ecd metabolites was determined by comparing their retention times with those of authentic standards (ecdysone. 20hydroxyecdysone, and makisterone A were obtained from Simes; ponasterone A and 22,25-dideoxyecdysone (trio]) were obtained from Dr. J. D. O’Connor; 20-hydroxyecdysonoic acid and 20,26dihydroxyecdysone were obtained from Dr. M. J. Thompson). Fractions from tissues incubated with [‘H]E were analyzed by scintillation spectrometry. Due to the extensive dilution of the small sample extracts by scintillation fluor, variations in counting efficiency were not significant. Fractions from unincubated MGs were analyzed by radioimmunoassay (RIA) (Chang and O’Connor, 1979) as previously described (Snyder and Chang, 1991a,b). The II-B Ecd antisera (obtained from Dr. W. E. Bol-

288

SNYDER

AND

lenbacher) was used in the RIA analyses. The antisera had a 2.3 x greater affinity for ecdysone than for either 20-hydroxyecdysone or ponasterone A. The data were not corrected for differential affinities. Recoveries of Ecds were approximately 85% as determined from inclusion of lsH]E in some tissue samples just before extraction. Enzymaric hydrolysis. Highly polar (HP) and apolar Ecd fractions from HPLC were evaporated under reduced pressure and hydrolyzed with a crude enzyme preparation (type H-2 Helix pomatia sulfatase; 10 mg/ ml; Sigma) in sodium acetate buffer (50 mM, pH S.5) at 37” for 48 hr. Methanolic extracts of the enzyme treatments were reanalyzed by RP HPLC followed by either scintillation spectrometry or RIA as outlined above. Prorein isolation. Lobsters were intubated with [3H]E (3.0 pCi) directly into the cardiac stomach as previously described (Snyder and Chang, 199lb). After 72 hr, lobsters were sacrificed and the MGs, midguts, and hindguts were removed, weighed, and homogenized in 5 vol of 0.1 M PO, buffer (pH 7.2) containing either 0.1 rni!4 PMSF alone or with the addition of 0.5 pg/ml leupeptin, 0.7 ug/ml pepstatin (both from Boehringer-Mannheim), and 1.0 mit4 sodium EDTA (Sigma). For studies of [3H]-labeled apolar Ecd association with protein, the homogenates were centrifuged at 100,OOOgfor 60 min and concentrated to 1.0 ml using a Centricon 10 microconcentrator (Amicon). The concentrates were loaded onto a Sephadex G-200 column (.1.6 X 39 cm) and eluted at 12.0 ml/hr with 0.1 M PO4 buffer at 4”. Fractions (2 ml) were collected and assayed for both total protein (Lowry et a/. , 195 1) and total radioactivity. In addition, portions of both the total extract and the fractions associated with the protein peak were subjected to RP HPLC followed by scintillation spectrometry for identification of Ecd metabolites. Fractions containing the peak of [3H] were collected, concentrated, layered (either fresh or frozen for up to 4 weeks after isolation) on lO-30% continuous sucrose gradients, and centrifuged at 82,000g for 16 hr. Each gradient was fractionated prior to scintillation spectrometry and total protein determinations. In addition, standard proteins (Bio-Rad) were included in some gradients for size determination of the apolar binding protein. Binding studies. Lobsters were intubated with either r3H]E or unlabeled E as outlined above. After 72 hr the MGs were removed, homogenized, extracted in methanol, and subjected to RP HPLC for the collection of apolar fractions. Measurements of recovered radiolabeled apolar Ecds were made by scintillation spectrometry of aliquots of the total. Unlabeled apolar fractions were pooled and subjected to Helix enzyme hydrolysis prior to RIA to obtain estimates of the total. Homogenates of MGs were prepared from intermolt stage C4 lobsters as outlined before. They were incu-

CHANG

bated with 3H-labeled apolar Ecds (0.8 r&f; 1.1 nCi) in the presence or absence of 400-fold excess unlabeled apolar metabolites for 2 hr at 4”. Bound apolar Ecds were separated from free by the addition of saturated ammonium sulfate in phosphate buffer. In other experiments, whole MGs were incubated with 3H-labeled apolar Ecds. Homogenates were then made and subjected to sucrose density gradient ultracentrifugation. Fractions were collected and analyzed as described above.

RESULTS

Midgut

Gland Ecdysteroids

Qualitative patterns of Ecd metabolites in MGs of juvenile lobsters in molt stages A, C,, D,, and D, were characterized using HPLC-RIA (Fig. 1). Tentative identification of Ecd metabolites was based on coelution (with authentic standards) from RP columns using different solvent systems. HP products (more polar than 20E) were observed to be the principal metabolites in all molt stages. The HP products comprised about 7&90% of the total RIA activity in the MGs of lobsters in all molt stages. Most of the HP products consisted of very polar (eluting first from RP HPLC) Ecd conjugates, with smaller quantities of 20hydroxyecdysonoic acid (20EA) and 20,26dihydroxyecdysone (2026E). The relative amounts of 20E and E in the MGs were always less than 10% of the total Ecds. Trace amounts (less than 2% of the total Ecds) of ponasterone A (P), intermediate polarity products (eluting between P and 22,2Sdideoxyecdysone, T), and low polarity products (eluting after T) were also found in the premolt stages D, and D, (Figs. lc and Id). Upon hydrolysis of crude MG extracts with H. pomatia sulfatase, the relative amounts of several Ecds increased in all samples (Fig. 1). Postmolt stage A RIA profiles after hydrolysis of crude MG extracts indicated that large amounts of 20E and P, smaller amounts of 20EA, and E, and a low polarity product were released. Intermolt stage C4 MGs had HP products that were

MIDGUT

GLAND

289

ECDYSTEROIDS

b

a

S!W 8W CYO hl* + ....f... ..

,I . =-iFi

'0

10

20

30

40

50

0

60

10

20

30

40

60

60

30

41 cl

:

C

.

,, !i 15..

0

la

20

30

40

60

60

0

d

.

.. ...... ..

l-

2

njjw

(Y

z:

10

20

.! ..n . :* . . .

+

30

40

60

60

Retention Time (min) FIG. 1. Patterns of ecdysteroids in the midgut glands of lobsters in molt stages A (a), C,(b), D, (c), and D, (d). Determinations were made by RIA of RP HPLC fractions of portions of midgut gland extracts (solid lines). The highly polar fractions (4-9) from these chromatograms were then collected and incubated with Helix pomatiu sulfatase (dotted lines). Symbols refer to highly polar Ecd conjugates (HPl) and the retention times for authentic standards of 20-hydroxyecdysonoic acid (20EA), 20,26-dihydroxyecdysone (2026E), 20-hydroxyecdysone (20E), ecdysone (E), ponasterone A (P). and 22,2Sdideoxyecdysone (T, Viol).

conjugates of roughly equivalent amounts of 2026E, 20E, and E with smaller quantities of intermediate polarity products. In the premolt stages, 20E conjugates were the principal HP products, while 2026E conjugates were reduced. Premolt stage D, also had detectable intermediate polarity product conjugates that were absent in late premolt stage D,. Small amounts of low polarity Ecds (coeluting with and after T), which were almost completely hydrolyzed following enzymatic treatment, were found in the premolt stages. Fractions 40-47 from RP HPLC containing the apolar Ecds were pooled, subjected to Helix sulfatase, and rechromatographed. Both 20E and E were the major Ecds released with slightly lower quantities of 2026E and traces of other metabolites. Less E was found as apolar conjugates in stage A

than in other molt stages. The total amount of Ecds released by hydrolysis, as determined by summation of RIA fractions after enzyme treatment and RP HPLC, is given in Fig. 2. All molt stages were significantly different from each other in terms of the total quantity of hydrolyzed Ecds per gram wet weight of MG in the following order: D, > A > D, > C,. When examined as a percentage of the total Ecds in the MGs, the order was D, > D, > A > Cd. The late premolt stage D, had the highest quantity of apolar Ecd conjugates (375 rig/g). This was at least 47% of the total Ecds in the tissue (Fig. 3). Although it was not determined whether enzymatic hydrolysis was carried to completion in all samples, these results clearly show that apolar conjugates were major components of the total Ecds in MGs at all molt stages.

290

SNYDER

A

C4 Molt

9

AND

D3

stage

FIG. 2. Amottnts of apolar Ecd conjugates found in the midgut glands of lobsters in molt stages A, C,, D,, and D,. Contents were determined by pooling RP HPLC fractions 4047 and subjecting them to Helix sulfatase hydrolysis followed by HPLC-RIA for quantification of the total released by hydrolysis (nanograms of Ecds released per gram wet wt of midgut gland). Bars represent the mean 2 one standard deviation of three or four determinations per molt stage. Data from each molt stage were significantly different from each other (P < 0.05).

Tissue Incubations Tissues from juvenile lobsters in molt stages C4 and D, were incubated with [‘H]E in saline. Table 1 indicates the rates of 13H]E metabolism by abdominal muscle,

CHANG

MG, epidermis, antenna1 gland, and hindgut. In stage C,, the order of highest to lowest [3H]E metabolic rate was antenna1 gland > epidermis = hindgut > MG > muscle; while in stage D,, the order was antenna1 gland > epidermis > hindgut = MG z muscle. The rate of [3H]E metabolism by antennal gland was more than twice the rate of any other tissue at either molt stage. Rates of [3HJE metabolism were significantly higher in both epidermis and antenna1 gland at stage D, as compared with stage C,. The patterns of 3H-labeled metabolites derived from [3H]E by in vitro incubations of stage C4 are shown in Table 2. At least two types of HP Ecd conjugates were found in all tissues, along with 20EA, 2026E, 20E, P, and intermediate products. Amounts of 3H-labeled HP were highest in hindgut and lowest in muscle. Levels of either [3H]20EA or [3H]2026E were never greater than 2.5% of the total [3H]Ecds in any tissue sample. The formation of [3H]20E was highest in epidermis, antenna1 gland, and hindgut. Levels of [3H]P were highest in muscle. Significant amounts of 3H-labeled apolar conjugates were produced only by the MG, although traces were also formed by epidermis and hindgut. Apolar Ecd-Protein

A

C4 Molt

D1

D3

Stage

FIG. 3. Quantities of apolar Ecd conjugates as percentages of the total Ecds in the midgut glands of lobsters in molt stages A, C,, D,, and D,. Hydrolyzed Ecds released from apolar Ecd RP HPLC fractions 40-47 were quantified by rechromatography and RIA analyses. Total amounts recovered were compared with those of the initial RP HPLC-RIA analyses of midgut gland methanolic extracts.

Studies

Lobsters were fed [3H]E, the MG was extracted 72 hr later, and the homogenate was separated on a Sephadex G-200 column. A large peak of radioactivity was found associated with a large protein peak that eluted shortly after the void volume (Fig. 4). Almost 60% of the [3H]Ecds were associated with less than 7% of the total protein in the MG homogenate. Methanolic extraction of a portion of the [3H]Ecd peak, followed by RP HPLC, indicated that it consisted of greater than 98% apolar Ecds. Hydrolysis of this material with H. pomatia sulfatase followed by RP HPLC confirmed that the 3H-labeled apolar material was principally a conjugate of E (ca. 96%) with

MIDGUT

[3H]E~~~~~~~

GLAND

291

ECDYSTEROIDS

TABLE 1 (fmol/mg wet wt . h) BY LOBSTER Tissuns IN VITRO

METABOLISM

Tissues” Molt stageb

C4

M

MG

EP

AG

HG

36 k 6”

117 f 44b Fns 149 f 51s

266 + 66’ k* 467 k 56’

564 k 158d I* 994 +- 109d

267 + 40’

hlS

,, 1Z-3

44k 11”

h-lS

229 f 3Sd

LITissues were rinsed in lobster saline (Mykles, 1980) and cultured with [3H]E for 12 hr at 20”. Both culture media and tissues were extracted in methanol, and portions analyzed by RP-HPLC followed by scintillation spectrometry. The total [3pH]E converted to other [3H]Ecd metabolites per hour was thus determined. M, abdominal muscle; MC, midgut gland; EP, epidermis; AG, antennal gland; HG, hindgut. Data represent the mean +- one standard deviation of three or four determinations. Signiticant differences (t tests, P < 0.05) between tissues within a molt stage are indicated by different superscript letters. Significant differences between the two molt stages for the same tissue are indicated by asterisks after the C symbols (P < 0.05). ’ Molt stages were determined according to the morphological designations of Aiken (1973).

only traces of P and HP products (data not shown). The fractions containing the peak of 3Hlabeled apolar Ecds, either fresh or frozen at - 20” for 8 weeks, were subjected to sucrose density gradient ultracentrifugation. TABLE

2

PATTERNS OF ECD METABOLITES IN STAGE LOBSTER TISSUES INCUBATED IN VITRO WITH

C, [3H]E

Tissue” Metabolite*

M

MC

EP

AG

HG

HP conjugates 20EA 2026E 20E P INT conjugates T Apolar conjugates Unmetabolized E

2.0 0.3 0.4 2.6 4.8 0.5 0.0 0.0 89.0

6.6 2.4 1.5 2.9 0.6 0.4 0.0 7.1 78.5

5.9 1.8 1.3 15.7 1.1 1.1 0.0 0.2 72.3

7.5 1.1 1.7 10.4 1.6 0.2 0.0 0.0 77.7

15.3 1.2 1.8 14.1 0.9 2.2 0.0 0.1 64.4

A large peak of 3H-labeled apolar Ecds (greater than 50% of the total) was associated with one of the five protein peaks in the gradients (Fig. 5). The protein had an approximate MW of 180,000 based on the sedimentation of protein standards. Incubation of the protein-associated 3H-labeled apolar Ecd fractions with XXI-fold excess 20E, E, or P at 21” for 2 hr failed to change the resultant sucrose gradient profiles (data not shown). Cytosolic extracts of unlabeled MGs were incubated with semipurified 3Hlabeled apolar Ecds in the presence or ab‘;‘ 20, 15

4

60

“,0

10

40

m 2 .;

5

20

% k F

0

2

E 02 ‘;;

a Tissue abbreviations are defined in Table 1. Data represent the mean of the percentages of total CPM (n = 2-3 determinations). ’ Metabolites were separated by RP HPLC and their tentative identifications were made based on coelution with authentic standards in at least two chromatographic systems (Snyder and Chang, 199la,b,c). Metabolite abbreviations are 2026E, 20,26-dihydroxyecdysone; 20E, 20-hydroxyecdysone; 20EA, 20hydroxyecdysonoic acid; E, ecdysone; HP, highly polar; P, ponasterone A; INT, intermediate polarity; and T, trio1 (22,25-dideoxyecdysone).

7 x b (L

0 0

10

20

30

40

50

60

Fraction

4. Elution profile from a Sephadex G-200 column of concentrated midgut gland cytosol from a lobster fed 72 hr previously with [3H]E. Total radioactivity and protein were monitored in lo-min fractions collected at a flow rate of 12.0 mUmin. The void volume (V,,) was determined by the retention time of Dextran Blue 2000. FIG.

292 7

SNYDER

20-

h,5y 6 0 tj Iz ‘; E b a

CHANG

2Or

-80

-

-60

T .!f,

-40

7

XI !,

AND

F lo-

01

d\

?

5--

2.

4 lb?&--

0

Bottom

5

0 10 Fraction

15

20

2 a’ -5 2

25 TOP

FIG. 5. Sucrose density gradient ultracentrifugation profiles of 3H-labeled apolar Ecds and total protein from Sephadex G-200 column fractions that contained a large peak of radioactivity associated with protein. Data points for cpm represent the percentage of the total radioactivity in the sample before ultracentrifugation. The abbreviations Th, Ga, Al, and Ov refer to the sedimentation positions of thyroglobulin (670 kDa) y-globulin (158 kDa), albumin (67 kDa), and ovalbumin (44 kDa), respectively.

0 sottom

5

10 Fraction

15

20

25 TOP

FIG. 6. Sucrose density gradient profiles of 3Hlabeled apolar Ecds from midgut gland cytosol incubated in the absence (tilled squares) or presence (open squares) of 400-fold excess cold apolar Ecds. Data points represent the percentage of each fraction of the total radioactivity in each treatment. Abbreviations for protein standards are as given in Fig. 5.

molting hormones 20E, E, and P were never at high levels in the MG at any molt stage. This evidence argues for an effkient sence of 400-fold excess unlabeled apolar metabolizing system that converts these Ecds (prepared in an analogous manner as Ecds to both HP and apolar conjugates. 3H-labeled apolar Ecds by direct feeding of The HP conjugates were previously identiunlabeled E into the cardiac stomach of jufied in lobster hemolymph (Snyder and venile lobsters). Sucrose density gradient Chang, 1991a,c) and urine (Snyder and centrifugation following the incubations Chang, 1991b,c), while the apolar conjushowed that the tritium was no longer gates were found only in lobster feces (Snypresent in those fractions from the middle der and Chang, 1991b,c). When crabs and of the gradient. A 250- to 400-fold excess of unlabeled Ecds reduced the association of lobsters were injected with [3H]E, the MG 3H-labeled apolar metabolites by 97.3 + concentrated more of the radiolabel than 1980, 1982; 1.2% (mean + one standard deviation of any other tissue (McCarthy, Snyder and Chang, 1991~). Similarly, the five determinations). The molecular weight MG of intermolt stage crabs, Pachygrapsus of the binding moiety was 180,00&200,000, crassipes, also concentrated more Ecds similar to that found for the binding protein than other tissues as determined by RIA isolated by gel filtration column chromatography (Fig. 6). Incubation of unlabeled cy- (Chang et al., 1976). The probable function tosol with 3H-labeled apolar Ecds with or of the MG in this regard is to absorb Ecds from the hemolymph and convert them into without IOOO-fold excess 20E, E, or P failed to change the density gradient profiles, sug- metabolites destined for excretion in the fegesting again that these metabolites did not ces (Snyder and Chang, 1991b,c). Another function of the MG is to compete for the same protein binding sites demonstrated transform ingested Ecds into apolar conju(data not shown). gates for fecal excretion (Snyder and Chang, 1991b), analogous to one of the DISCUSSION functions of the gut in other arthropods Ecdysteroid Profiles and Metabolism (Isaac and Slinger, 1989). MG Ecd profiles varied over the four lobWhen RP HPLC specific fractions of ster molt stages. The combined “active” MGs containing little or no RIA activity

MIDGUT

GLAND

(fractions with 4U7 min retention time; Fig. 1) were collected and subjected to Helix enzyme hydrolysis, significant amounts of Ecds appeared upon reanalysis by HPLC-RIA. These apolar Ecds were also formed by MGs incubated in vitro with [3H]E (Table 2). Others have reported the presence of apolar Ecds in crayfish MG and integument (Gore11 and Gilbert, 1972; Gore11 et al., 1972; Kuppert et al., 1978; Durliat et al., 1988), shrimp integument (Baldaia et al., 1984), shrimp embryos (Spindler et al., 1987), and crab larvae (Connat and Diehl, 1986). These apolar Ecds may be widespread in crustaceans and more research will be necessary to delineate their function(s). In other arthropods, apolar Ecd conjugates are associated with Ecd excretion in adults (Connat et al., 1988) and pupae (Delbecque et al., 1988) or serve as possible Ecd-storage forms for developing embryos (Slinger and Isaac, 1988; Whiting and Dinan, 1988). In lobsters, concentrations of MG apolar Ecds vary with the molt cycle (Figs. 2 and 3). The highest levels were found in late premolt (stage D3) and postmolt (stage A). These data suggest that apolar Ecd conjugates may be stored faster than they are excreted in the feces. As a consequence, stage A apolar Ecd content was higher and dropped through late postmolt-early intermolt to the lowest values found in stage C,. Alternatively, some of the MG apolar Ecds could be hydrolyzed to free Ecds which diffuse back into the hemolymph. Lobsters have a small transitory hemolymph Ecd peak (consisting of ca. 75% HP Ecds) in late postmolt-early intermolt (Snyder and Chang, 1991a). It is possible that this small peak could arise from the loss of Ecds stored by tissues, such as MG, during late premolt when production far exceeds excretion rates. There is currently insufficient information to determine whether any of these apolar Ecd conjugates could be reutilized in this manner. Some evidence has been obtained that suggests that enough

ECDYSTEROIDS

293

Ecd is stored in the insect pupal midgut to drive the pupal-adult transition (Williams, 1987). Apolar Ecd conjugates were also formed by MGs in culture with [3H]E. Of the four additional types of tissue cultured in vitro, only epidermis and hindgut produced traces of 3H-labeled apolar Ecds (~0.2%). Crayfish MG, but not abdominal muscle, produced apolar Ecds when cultured for 5 hr with [3H]E (Gore11 and Gilbert, 1972). All of our lobster tissues produced 20E and more polar products that cochromatographed with 2026E, 20EA, and HP conjugates. These data confirm the suggestion that most crustacean tissues generate HP Ecd metabolites which are probably destined for excretion in the urine (Snyder and Chang, 1991b,c). Highly polar Ecds are also produced by cultured tissues of the crayfish Orconectes virilis (Gore11 and Gilbert, 1972) and the crab Carcinus maenas (Lachaise and Feyereisen, 1976; Lachaise and Lafont, 1984). The increased hydroxylation, followed by the formation of acids and HP products, has previously been reported to be the primary pathway for crustacean Ecd inactivation (McCarthy, 1980, 1982; Lachaise and Lafont, 1984; Snyder and Chang, 1991a,b,c). In light of our data on lobster Ecds, perhaps the apolar Ecd metabolic pathway is equally important. Antenna1 glands were the most active [3H]E-metabolizing tissue in culture. Its metabolizing activities were 2- to lo-fold higher than those in other tissues. The MG activity was generally intermediate in activity between the abdominal muscle and the antennal gland. However, due to its large volume (ca. 5% of the total wet wt) relative to other tissues, the MG was the most active [3H]E-metabolizing tissue. These results confirm earlier data from [3H]E injection studies in which the MG concentrated more radiolabel than other tissues (Snyder and Chang, 1991~). The [3H]E-metabolizing activity nearly

294

SNYDER

AND

doubled in the antenna1 gland and the epidermis between stages C4 and D,. Similarly, Chang and O’Connor (1978) found that ecdysone 20-monooxygenase activity increased 3-fold in testis of the crab, P. crussipes, following molt induction using bilateral eyestalk ablation. There was no change in lobster MG activity in premolt stage D,. James and Shiverick (1984) determined that ecdysone 20-monooxygenase activity was nearly 60-fold higher in the antennal gland than in the MG in the spiny lobster, Panulirus argus. It is possible that in lobster the E-metabolizing activity in both the antenna1 gland and the epidermis may be regulated during the molt cycle. When lobsters were injected with [3H]E, the rates of metabolism and hemolymph clearance of [3H]E and [3H]20E varied with the molt stage. The half-life of [3H]20E, for example, was longest in the premolt stages. However, in late premolt stage D,, a much shorter half-life for [3H]20E was associated with the rapid decline in the hemolymph ecdysteroid titer seen just prior to ecdysis (Snyder and Chang, 1991a,c). Both the in vivo evidence and the in vitro evidence in lobsters suggest that the control of hormone metabolism itself may be an important mechanism for the regulation of circulating Ecd titers. Similarly, Soumoff and Skinner (1988) found that E-20-monooxygenase activity in crab MG was lower in very late premolt and postmolt. Apolar Ecd-Protein

Associations

Lobsters fed [3H]E absorbed the label into the MG and converted it principally to 3H-labeled apolar metabolites. The apolar Ecds became associated with a cytosolic protein(s) that could be characterized by gel filtration column chromatography and sucrose gradient centrifugation analyses. The binding was significantly reduced by incubation in the presence of excess unlabeled apolar Ecds, but not by excess 20E, E, or P (Fig. 6). The specificity of the pro-

CHANG

tein(s) for apolar Ecds was demonstrated by the failure of 20E, E, or P to compete for binding. These results are similar to those reported from in vitro culture of crayfish, 0. virilis, MG with [3H]E (Gore11 and Gilbert, 1972; Gore11 et al., 1972). The component bound to crayfish protein was less polar than P in thin-layer chromatography, but was not further identified. The binding protein was not found in crayfish abdominal muscle. In the lobster assays, 20E, E, and P were not able to compete with apolar metabolites for association with this protein. Therefore, the binding protein is probably not the ecdysteroid receptor which has been found in crayfish MG (Spindler-Barth et al., 1981; Londershausen and Spindler, 1985). Additional evidence supporting the idea that the ecdysteroid receptor and the MG binding protein are different is the observation that the apolar binding moiety has a molecular weight estimated at >180,000. The molecular weight of the ecdysteroid receptor from the crayfish MG was estimated at 30,000-80,000 by photoaffinity labeling (the smaller component was assumed to be a degradation product of the larger one) (Londershausen and Spindler, 1985). The question of the function of the apolar binding protein in the decapod crustacean MG remains. It is possible that this protein may function in a manner analogous to that of the vertebrate binding proteins which are responsible for intracellular transport of fatty acids (Kaikaus et al., 1990). The lobster apolar Ecd conjugates have not yet been further characterized, but all such molecules have thus far been identified as long-chain fatty acid esters in other arthropods (Kubo et al., 1987; Whiting and Dinan, 1989). Potentially, the crustacean apolar Ecd binding protein may be involved in the directed transport of these metabolites to the lumen of the gut for excretion in the feces. Alternatively, the protein could serve as a storage reservoir for these metabolites that may, by hydrolysis, be reuti-

MIDGUT

lized in their active forms following port to the hemolymph.

ECDYSTEROIDS

trans-

brate Types” (H. Laufer and R. G. H. Downer, Eds.), Vol. 2, pp. 259-288. A. R. Liss, New York. Chang, E. S., Sage, B. A., and O’Connor, J. D. (1976). The qualitative and quantitative determinations of ecdysones in tissues of the crab, Pachygrapsus crassipes, following molt induction. Gen. Comp. Endocrinol. 30, 21-33. Cheng, J.-H., and Chang, E. S. (1991). Ecdysteroid treatment delays ecdysis in the lobster, Homarus americanus. Biol. Bull., 181, 169-174. Conklin, D. E., and Chang, E. S. (1983). Grow-out techniques for the American lobster, Homarus americanus. In “CRC Handbook of Mariculture” (J. P. McVey, Ed.), Vol. 1, pp. 277-286. CRC Press, Boca Raton. Connat, J. L., and Diehl, P. A. (1986). Probable occurrence of ecdysteroid fatty acid esters in different classes of arthropods. Insect Biochem. 16,9197. Connat, J. L., Fihst, P. A., and Zweilin, M. (1988). Detoxification of injected and ingested ecdysteroids in spiders. Camp. Biochem. Physiol. B 91, 257-265. Daig, C., and Spindler, K.-D. (1983). Uptake and retention of moulting hormones by the integument of crayfishes in vitro. II. Influence of metabolic inhibitors and sulphydryl group inhibitors. Mol.

ACKNOWLEDGMENTS We thank Dr. W. E. Bollenbacher (University of North Carolina, Chapel Hill) for the generous gift of ecdysteroid antisera, and Dr. M. J. Thompson (U.S. Department of Agriculture, Beltsville, MD) for several ecdysteroid standards. We also thank Drs. W. H. Clark and B. L. Lasley for review of the manuscript, and Dr. J.-H. Cheng for helpful discussions. This work is a result of research sponsored in part by NOAA, National Sea Grant College Program, Department of Commerce, under Grant NA85AA-D-SG140, Project R/A-80, through the California Sea Grant College Program (to E.S.C.). The U.S. Government is authorized to reproduce and distribute copies for governmental purposes.

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