estradiol induces proliferation of peroxisome-like microbodies and

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 15, Issue of May 25, pp. 9795-3804,1991 Printed in (1. S. A.

Estradiol Induces Proliferation of Peroxisome-like Microbodies and the Production of 3-Hydroxy Fatty Acid Diesters, the Female Pheromones, in the Uropygial Glands of Male and Female Mallards*

(Received for publication, May 17, 1990)

Stewart Bohnet, Linda Rogers, Glenn Sasaki, and Pappachan E. KolattukudyS From the Ohio State Biotechnology Center, The Ohio State University, Columbus, Ohio 43210

During the mating season the female mallards produce sex pheromones, diesters of 3-hydroxy fatty acids, in their uropygial glands. Subcellular fractionation by sucrose and Nycodenz density gradient cen- trifugations and electron microscopic examination of the fractions showed that diesters of 3-hydroxy acids and the enzymes that catalyze the formation and esterification of the 3-hydroxy fatty acids are located in the catalase-containing fractions, probably peroxisomes, whereas monoester synthesizing activities are located in the endoplasmic reticulum. Fatty acyl-CoA reductase that would provide fatty alcohol needed for the synthesis of monoester and diester waxes was found both in the peroxisomal and endoplasmic reticulum fraction. Upon daily intramuscular injection of estradiol into the females in the nonmating season, the short chain monoester waxes of the uropygial glands were replaced by long chain monoester waxes, and subse- quently the monoester waxes were replaced by diester waxes. Injection of thyroxine with estradiol hastened the induction of the compositional changes including diester synthesis. Similar changes, including the synthesis of the female pheromones, were induced in the uropygial glands by the hormone treatment of males that do not normally produce diesters at any time during their life cycle. The structure and composition of the diesters induced by hormone treatment of both males and females were identical to those of the female pheromones produced during their mating season. Electron microscopic examination of diaminobenzidine-treated glands showed that peroxisomes prolif- erated in the gland of the females in the mating season and in the estradiol-treated males that produce the diesters.

Sebaceous glands produce unusual lipids that perform a variety of functions (1). The structure and function of the diminutive mammalian sebaceous glands that are widely dis- tributed on the animal surface are quite similar to those of the single avian sebaceous gland called the uropygial gland. Therefore, the avian gland is a convenient model to elucidate

* This work was supported in part by Grant GM-18278 from the National Institutes of Health. The work was initiated at the Institute of Biological Chemistry a t Washington State University, Pullman, WA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed Ohio State Bio- technology Ctr., The Ohio State University, 206 Rightmire Hall, 1060 Carmack Rd., Columbus, OH 43210.

the biochemistry and molecular biology of the sebaceous gland.

The composition of the lipids produced by sebaceous glands has been suggested to be useful as a chemotaxonomic marker (2). However, little attention has been paid to the possibility that the composition might be determined by the physiological and hormonal factors that regulate the metabolism of sebaceous glands. Such a case was first noted when age-dependent changes in the diastereoisomer composition of the uropygial gland lipids of chickens were examined (3). During eclipse, the period immediately following postnuptial molt of mallard ducks, esters of short chain fatty acids that normally constitute S O % of the secretion are replaced by esters of long chain acids (4). This change was shown to be caused by suppression of the expression of the gene for the thioesterase responsible for releasing short chain acids from fatty acid synthase ( 5 ) . A dramatic change in the composition of the sebaceous gland secretion was found in the female mallards during the mating season. During this 2-month period, the usual monoester wax was completely replaced by diesters of 3-hydroxy fatty acids (6) that have been reported to be duck pheromones (7). Biosynthetic studies on the 3-hydroxy fatty acids showed that peroxisomal enzymes are involved in the synthesis of these acids (8). How the sebaceous gland switches its biochemical processes to produce the pheromone is not known. In this paper, we show that injection of estradiol into mallards induces the uropygial glands to switch from the synthesis of the usual monoester waxes to the diesters not only in the females that normally undergo such a change in the mating season but also in the males that do not normally make diesters a t any time. Estradiol treatment during the nonmating period is shown to induce peroxisome proliferation in the sebaceous gland. Hormonal induction of peroxisome proliferation has not been reported heretofore. Peroxisome proliferation was also observed in the gland during the mating season when the females naturally produce the diesters. Thus the biochemical and ultrastructural studies suggest a new biosynthetic role for peroxisomes.

EXPERIMENTAL PROCEDURES

Materials-Mallard ducks (Anas plutyrhynchos), 8 months old and weighing about 1 kg each, were purchased from Robert Cramer (Centralia, WA) or from Whistling Wings (Hanover, IL) and maintained in outdoor cages on a high energy breeder ration. Lipid secretion was obtained by gently squeezing the uropygial gland, and the lipid samples were stored at -20 “C until further workup.

Sodium L-thyroxine, 17P-estradiol benzoate, olive oil, 14% boron trifluoride in methanol, N,O-bis(trimethylsilyl)acetamide, NADPH, NADH, ATP, CoA, palmitoyl-CoA, bovine serum albumin, and dithioerythritol were purchased from Sigma. Nycodenz was purchased from Accurate Chemical and Scientific Corporation (Westbury, NY). Hexadecanol and 3-hydroxy Cla and C12 acids were purchased from Analabs (North Haven, CT). ‘H20 (63 Ci/mol), [1-“C]dodecanoic

9795

9796 Estradiol Induces Peroxisomes and Diesters in Mallards acid (50-60 Ci/mol), and [l-'4C]hexadecanoic acid (50-60 Ci/mol) were purchased from Amersham Corp.

[l-'4C]Dodecanoyl-CoA (59 Ci/mol), [1-'4C]hexadecanoyl-CoA (53 Ci/mol), and [l-14C]hexadecanol (58 Ci/mol) were prepared as previously described (9). 3-Hydro~y[2-~H]decanoic acid was produced by the butyl1ithium:diisopropylamine-mediated exchange of 3H from "Hz0 to methyl 3-hydroxydecanoate (10). The methyl ester was subjected to base hydrolysis and the hydroxy fatty acid was purified by thin layer chromatography on Silica Gel with ethyl ether: hexane:methanol:formic acid (40:10:1:2, v/v) as the solvent.

Hormonal Injection-Birds were divided into four groups for male and female ducks (four per group) to receive injections of sodium L- thyroxine (T4 only),' 170-estradiol benzoate (E only), sodium L- thyroxine plus 170-estradiol benzoate (T4/E) or olive oil only (control). During the period from late November to early February, ethanolic solutions of hormones, usually 1 mg each, were mixed with a 19-fold volume of olive oil, and the mixture was injected intramus- cularly into the flight muscle of each bird daily. The olive oil and ethanol mix alone was used for treatment of control ducks.

Analysis of Wax Samples-The crude wax samples were subjected to thin layer chromatography on 1-mm-thick Silica Gel G with a 901O:l (v/v) mixture of hexane, ethyl ether, and formic acid as the developing solvent. Components were visualized under UV light after spraying the plates with a 0.1% ethanolic solution of 2,7-dichlorofluorescein. Wax ester and diester fractions were recovered from Silica Gel by elution with diethyl ether. Thin layer chromatographic plates were also sprayed with 0.5% K2Cr04/50% H2S04 and charred at 180 "C for 10 min to visualize individual bands. A Hewlett Packard model 5840A gas chromatograph attached to a HP 5985 mass spectrometer was used to obtain electron impact mass spectra of the intact waxes. A glass capillary column (10 m X 0.2 mm, OV-1) was used with a 1- min isothermal period at 150 "C followed by a 150-280 "C temperature program of 10 "C per min. Mass spectra were recorded with 70 eV ionizing voltage.

Compositional Analysis of 3-Hydroxy Fatty Acid Diester Waxes-A 2-4-mg sample of diester wax from the hormone-treated animals was dissolved in 0.5 ml of toluene, 3 ml of 14% BF, in methanol was added, and the reaction mixture was refluxed for 3 h. The reaction products were mixed with 10 ml of water, the products were extracted with chloroform, and the solvent was removed under reduced pres- sure. The residue was subjected to thin layer chromatography on Silica Gel G with hexane:ethyl ether:formic acid (90:10:1, v/v) as the solvent. The fatty acid methyl esters and the fraction containing both the 3-hydroxy fatty acid methyl esters and fatty alcohols were recovered from the Silica Gel by elution with ethyl ether and chloroform:methanol (2:1), respectively. The alcohol-hydroxy acid methyl ester fraction was treated with N,O-bis(trimethylsily1)acetamide for 20 min at 90 "C before subjecting it to gas chromatography. Areas were calculated from the flame ionization detector response following separation on a glass capillary column (25 m X 0.2 mm, OV-1) with a 1-min isothermal period at 150 "C followed by a 150-280 "C temperature program at 10 "C per min. Mass spectra were used to identify the components as done previously (6).

Sucrose Density Gradient Isolation of Peroxisomes-The gland from a female mallard producing the diester during the mating season was carefully excised (11) and weighed. The tissue was finely chopped and homogenized in 20 ml of 10 mM HEPES, pH 7.4, containing 1 mM EDTA, 0.25 M sucrose, and 0.1% ethanol. The homogenate was centrifuged for 10 min at 1000 X g. The supernatant was centrifuged at 105,000 X g for 30 min. The resulting pellet, containing about 200 pg of protein, was gently resuspended in 1 ml of homogenization buffer and carefully layered on top of the following sucrose step gradient containing 10 mM HEPES, pH 7.4, 1 mM EDTA, and 0.1% ethanol: 5 ml each of 20, 28, 34, and 40% sucrose solutions, 2.5 ml each of 42,44,46, and 48% sucrose solutions with a 2-m160% sucrose cushion. The gradient was placed in a SW-28 rotor and centrifuged at 76,000 X g for 30 min at 4 "C. Fractions of approximately 1.75 ml each were collected from the bottom and placed on ice until analysis of marker enzymes.

Nycodenz Density Gradient Isolation of Peroxisomes-Homogenate from uropygial gland tissue that was exclusively producing diester was prepared and centrifuged at 1000 X g as described in the sucrose density gradient fractionation. The supernatant (2.5 ml) was layered

'The abbreviations used are: T4, sodium L-thyroxine; E, 170- estradiol; GLC/MS, gas-liquid chromatography/mass spectrometry; SID, selection ion display; HEPES, 4-(2-hydroxyethyl)-l-piperazine- ethanesulfonic acid ER, endoplasmic reticulum.

on a continuous Nycodenz gradient (12) and centrifuged 70 min at 130,000 X g (average) in a Beckman VTi-50 rotor. Fractions (1.2 ml each) were collected from the bottom of the tube, and aliquots were assayed for marker enzyme activity. Selected fractions were then assayed for diester formation and P-hydroxylation, and portions of these fractions were processed for electron microscopic examination. The lipids from the particles recovered from the fractions were extracted with a 2:l mixture of chloroform and methanol, and the diester fractions isolated by thin layer chromatography were subjected to GLC/MS; quantitation of diesters was done by SID using a representative ion at m/e 143, characteristic of the diester (see Fig. 3). Arbitrary units of ion intensity were used for comparison.

Enzyme Assays-Mitochondrial cytochrome-c oxidase was measured by the decrease in absorbance at 550 nm at 37 "C according to the method of Peters et al. (13). Cytochrome c was reduced with 6 mM sodium dithionite. Each assay mixture contained 0.5 mg/ml reduced cytochrome c in 0.1 M sodium phosphate, pH 7.0, containing 0.1% Triton X-100 and 1 mM EDTA in a total volume of 1 ml. Cytochrome-c reductase was measured spectrophotometrically at 25 "c (14). Each assay was in a total volume of 0.5 ml of 0.05 M potassium phosphate, pH 7.4, containing 0.1 mM NADPH, 0.1 mM cytochrome c and 0.3 mM KCN. Catalase was measured as described before (13). Diluted enzyme preparations were incubated at 25 "C in 0.1 ml of 0.02 M imidazole buffer, pH 7.0, containing 0.2% (w/v) Triton X-100 and 0.06% H202. Remaining hydrogen peroxide was estimated by the addition of titanium peroxysulfate. Fatty acyl-CoA oxidase was measured spectrophotometrically as described before (15). To measure acyl-CoA reductase activity, aliquots of the resuspended high speed pellet (10, 20, and 40 gl) and of each gradient fraction (300 pl) were assayed with 4 mM NADPH, 4 mM NADH, 0.5 mg of bovine serum albumin, and 250 p~ [l-'4C]palmitoyl-CoA (2 Ci/mol) in a total volume of 0.5 ml of 0.1 M sodium phosphate buffer, pH 6.5, containing 1 mM dithioerythritol. After incubating the mixture for 30 min at 30 "C, 50 pl of 6 N HC1 was added, and the primary alcohol product was isolated and quantitated as previously described (9).

To measure wax ester formation by the sucrose density gradient fractions, a 0.5-ml reaction mixture containing an aliquot of the resuspended high speed pellet (10, 20, and 40 p1) or gradient fraction (200 pl), 50 p~ palmitoyl-CoA, and 150 p~ [l-'4C]hexadecanol (7 Ci/ mol) in 0.1 M sodium phosphate buffer, pH 7.4, containing 1 mM dithioerythritol was incubated for 30 min at 30 "C. The labeled wax ester was isolated and quantitated as previously described (9).

To measure 0-hydroxylation by the sucrose density gradient fractions, a 0.5-ml reaction mixture containing an aliquot of the resuspended high speed pellet (5, 10, 20, and 40 p1) or gradient fraction (125 pl), 0.8 mM ATP, 0.05 mM CoA, 0.25 mg of bovine serum albumin, and 500 ~ L M [l-'4C]lauroyl-CoA (1 Ci/mol) in 0.1 M sodium phosphate buffer, pH 6.5, containing 1 mM dithioerythritol was incubated for 30 min at 30 "C. The products were analyzed as previously described (8).

To measure diester formation by the sucrose density gradient fractions, a 0.5-ml reaction mixture containing an aliquot of the resuspended high speed pellet (10, 20, 40, and 80 pl) or gradient fraction (200 pl), 0.8 mM ATP, 50 pM CoA, 50 g M lauroyl-CoA, 50 p~ hexadecanol, and 3-hydro~y[~H]decanoic acid (1 Ci/mol) in 0.1 M sodium phosphate buffer, pH 7.4, with 1 mM dithioerythritol was incubated for 30 min at 30 "C. The reaction was stopped by the addition of 50 pl of 6 N HCl, and the lipids were extracted by the method of Folch et al. (16). An aliquot of the lipid was assayed for total recovered radioactivity, and 50% of the recovered lipid was mixed with authentic diester isolated from mallard uropygial gland exudate as the internal standard and subjected to thin layer chromatography on 0.5-mm-thick Silica Gel G with hexane:ethyl ether:formic acid (9010:1, v/v) as the developing solvent. The diester area was visualized under UV light after spraying the plate with a 0.1% ethanolic solution of 2,7-dichlorofluorescein, and the Silica Gel from this region was scraped into a scintillation vial and assayed for radioactivity.

To measure diester formation and @-hydroxylation from the Ny- codenz density gradient fractions, two-thirds of each fraction was diluted 5-fold with the homogenization buffer and centrifuged 30 min at 105,000 X g. Each pellet was resuspended in 300 p1 of homogenization buffer and half of this suspension was assayed for diester formation as indicated above, except that 300 p~ [l-'4C]dodecanoic acid (3.3 Ci/mol) was used instead of the labeled 3-hydroxydecanoic acid. Assay for 0-hydroxylation was done using the same reaction mixture except that hexadecanol was omitted and total 3-hydroxy

Estradiol Induces Peroxisomes and Diesters in Mallards acid formed was measured as previously described (8).

Protein was quant,itated by the method of Lowry et al. (17). Sucrose concentration in the gradient fractions was measured by refractive index. S-Acyl fatty acid synthase thioesterase level was measured by the immunoblot procedure (18) with rahbit IgG prepared against the thioesterase purified from the uropygial glands of mallards (19).

Ultrastructure-The female mallards during the mating season and hormone-treated males in the nonmating period, hoth groups producing diesters, and females during nonmating season and control males, hoth groups producing wax esters, were anesthetized with ketamine and xylazine. Since the caudal artery and vein are not readily accessible for perfusion, aortic puncture through the heart was used for perfusion of the whole animal. First, saline solution (0.9% NaCI) was used to remove erythrocytes, followed by 2.5% gluteraldehyde in cacodylate buffer (0.1 M, pH 7.2, containing 4% sucrose). The uropygial gland was removed and thin slices from the gland were further fixed in cacodylate-buffered 2.5% gluteraldehyde for an additional 5 h a t 40 "C. The slices were rinsed three times in cacodylate buffer, placed in peroxisome incubation medium (20) for 2 h a t 37 "C and rinsed again three times in cacodylate buffer. The slices were postfixed in 10; osmium tetroxide in cacodylate buffer for 1 h a t 4 "C, rinsed three times, and stored overnight in cacodylate buffer at 4 "C. The tissue was then dehydrated with a graded series o f ethanol (50-100%) and placed in propylene oxide twice for 5 min each. The samples were infiltrated with spur resin and then em- bedded. Thin sections, approximately 70 nm thick, were cut on a Heichert Ultracut E. Sections were stained in Reynold's lead citrate for 3 min. dried, and photos were taken with a Phillips 300 electron microscope.

Nycodenz gradient fractions were diluted 5-fold with the homogenization buffer and centrifuged 30 min a t 105.000 X ,q. The sucrose density gradient fractions were diluted to 0.25 M sucrose and centrifuged at 105,000 X g. The resulting pellets were fixed for 30 min with Xrh gluteraldehyde in 0.1 M cacodylate buffer, pH 7.4, with 4% sucrose and then treated by the same postfixation procedure as was used for the tissue slices. Many sections representing various regions of the pellets were examined and a representative segment is presented (see Fig. 6). The summary of the ohservations is given under "Results."

In a separate experiment, the Nycodenz fractions containing diester-formation activity were pooled and divided into three equal aliquots, which were diluted, centrifuged, and gluteraldehyde-fixed as described. The fixed pellets were incubated with the diaminohenzidine-containing peroxisome incubation medium as described except that in one case the incubation mixture also contained 20 mM 3- amino-1,2,4-triazole and in another 2 mM sodium cyanide.

RESULTS

Hormone-induced Changes in the Thin Layer Chromato- graphic Pattern of Uropygial Gland Lipids-Thin layer chromatographic analysis of the uropygial gland lipids collected from female mallards in the nonmating season, injected with &estradiol (E only) and sodium t-thyroxine with /3-estradiol (T,/E), showed that the hormone treatments caused changes in the lipid composition similar to that which occur during the mating season. The thin layer chromatography of uropygial gland lipids of normal female mallards synthesized during December and January showed a single band consisting of wax esters of short chain fatty acids (4). Following daily estradiol injections, a second more nonpolar component with a RF similar to that of the long chain monoesters and a third more polar component with a RF similar to that of the diesters of 3-hydroxy fatty acids appeared (E only, Fig. 1). Treatment with both estradiol and thyroxine hastened the changes in the composition; the new nonpolar component appeared by the 8th day, and the more polar component was found by the 10th day (TI/& Fig. 1). With either estradiol alone or estradiol with thyroxine, the polar diester component became the only component of the secretion as previously seen during the mating season. Injection of olive oil and thyroxine alone did not produce any change in the thin layer chromatographic pattern of the lipids. Following cessation of hormonal injections the composition of the gland secretion, as revealed by the thin layer chromatographic pattern, reverted back, first

FEMALE

9797 MALE

2 8 10 12 14 16 20 24 28

DAY

2 46.m 0 W 0

2 8 10 12 14 16 20 24 28 32 DAY

FIG. 1. Th in l aye r ch romatograms of the uropygial g land secret ions of male and female mal lards during a regimen of hormone inject ion as described in the text. The top rfouhld of hands is composed of monoester waxes and the louwr hand represents diesters of 3-hydroxy fatty acids. The hormone injections into the females were discontinued on days 14 and 20 for thyroxine/l:,j- estradiol (",/E) and 17Lj-estradiol ( E 0nl.v). respectively.

to long chain esters and then to short chain esters, as seen in the natural cycle of the female.

Estradiol injection into male mallards also caused compositional changes similar to those observed with females. Ry day 12, the more nonpolar component corresponding to long chain monoester wax appeared, and by day 20 the more polar diester-like component began to appear (Fig. 1). As observed with the females, thyroxine hastened the compositional changes. For example, the more nonpolar monoester wax appeared by day 8 and the polar diesters appeared bv dav 14 (Fig. 1). Either with estradiol alone or with thyroxine and estradiol, the more polar diester became the only component of the secretion of the uropygial gland. Dailv injections of estradiol, either alone or with thyroxine, maintained the diesters as the only component for 2 months (data not shown). Injection of olive oil and thyroxine alone did not cause detectable changes in the composition.

Identification of the Diesters Produced as a Result of Hor- mone Treatment-The polar component was examined by treatment with methanol containing 14% RFs followed by thin layer chromatographic analysis. Three components were found, one with a RF equal to that of fatty acid methyl esters and the other two having RF values equal to those of hexadecanol and methyl 3-hydroxydecanoate. The last two were isolated together and analyzed by gas-liquid chromatography and mass spectrometry of their trimethylsilyl derivatives. Of the five components, the three that emerged first were identified by the mass spectra to be 3-hydroxy CR, Cln, and C12 acid methyl esters, as described before (6). The two components with longer retention times were identified as hexadecanol and octadecanol by their mass spectra. The fatty acids were determined to be even numbered n-C6-C16 homologues. The composition of diesters of 3-hydroxy fatty acids collected from female birds injected with estradiol alone or with thv- roxine and estradiol matched with that isolated from females during the mating season (Table I). The Composition of the diesters produced by hormonal injections of the male birds was identical to that produced by the females (data not shown).

2 8 10 12 14 16 20 24

DAY

9798 Estradiol Induces Peroxisomes and Diesters in Mallards TABLE I

The composition of the fatty acids, 3-hydroxy fatty acids, and primary alcohols of the natural diester wax in the mating season and

estradiollthyroxine-induced diester wax from the uropygial glands of female mallards.

Component Estradiol Mating season

thyroxine (may 21) % of total

Fatty acid Hexanoic 2.4 5.1 Octanoic 9.8 7.2 Decanoic 33.7 32.6 Dodecanoic 20.6 21.2 Tetradecanoic 15.7 12.0 Hexadecanoic 15.7 17.8 Octadecanoic 2.1 4.1

3-Hydroxy fatty acid Octanoic 11.9 13.3 Decanoic 69.3 68.3 Dodecanoic 18.8 18.4

Fatty alcohol Hexadecanol 45.6 47.1 Octadecanol 54.4 52.9

ND" 13.6 37.6 23.9 12.4 12.5 ND

17.8 68.8 13.4

43.5 56.5

ND, not detectable by GC.

Capillary gas-liquid chromatograms of the intact lipids collected from male mallards 2, 12, and 20 days after the beginning of daily injections with estradiol and thyroxine showed distinct patterns suggesting three types of waxes (Fig. 2). Chromatographic analyses of lipids from females indicate that the females responded to hormone injection in a manner similar to the males (data not shown). To determine compositional changes in the wax esters and diesters induced by hormonal treatment, these lipids were subjected to gas-liquid chromatography and mass spectrometry. The fatty acid chain length of the wax ester portion was identified by the diagnostic ion representing the fatty acid portion (RCOH;) (21,22). The percentage of the total fatty acids for each chain length was calculated with the selected ion display (SID) option of the mass spectrometer. This allows for the simultaneous display and quantitation of selected ion profiles derived from the total ion data. All values were standardized to the percentages obtained with equimolar amounts of octadecyl heptanoate and octadecyl octadecanoate wax esters and authentic 3- hydroxy fatty acid diesters. The base peak of the mass spectra of the intact diester corresponds to the 3-hydroxy fatty acyl moiety as illustrated by the spectrum in Fig. 3. In this spectrum of the diester of 3-hydroxy Cs acid esterified to n-CI6 alcohol and Cs acid, a small molecular ion is seen at mle 482. The other major ions are probably generated in the following manner: a-cleavage removal of hexanoyl moiety yielded a major ion at mle 383 and elimination of hexanoic acid yielded an ion at mle 366. 3-Hexanoyloxyoctanoic acid with an additional hydrogen from the hydrocarbon chain of the alcohol generated an ion at mle 259. a-Cleavage removal of hexade- coxy moiety yielded an ion at mle 241. Elimination of the hexadecyl moiety with transfer of hydrogen to the carboxyl oxygen would generate an ion at mle 159 from the ion at ml e 383. The base peak at mle 143 represents elimination of hexanoic acid from the molecular ion followed by elimination of hexadecyl moiety with transfer of 2H' to the carboxyl oxygens, a major cleavage pattern usually observed in the spectra of wax esters. Therefore, the base peak reveals the chain length of the 3-hydroxy acid component of the diester; the ions at mle 143, 171, and 199 would represent 3-hydroxy acyl Cs, Clo, and CI2, respectively. The percentage composition of 3-hydroxy fatty acids calculated with the SID using ions at

- 5 10 TIME (MIN)

FIG. 2. Gas chromatograms of intact lipids produced by the uropygial glands of male mallards injected with thyroxine and estradiol for the number of days indicated on each tracing. Top, short chain esters; middle, long chain esters produced after 12 days of hormone treatment; bottom, 3-hydroxy fatty acid diesters found after 20 days of hormone treatment.

(Ie0 1 143

125 1 159 241

259

203 224

60 00 I00 120 140 160 180 200 220 240 260 ::I 4 0 366

I 383 20i 295 313 326 339 411 426 446 482

0 , . , , , . I . , . , . I . , . , . , . I ~ , . I ~ . . , , . , . , . , , . , . I 280 300 320 340 360 380 400 420 440 460 480 600

FIG. 3. Mass spectrum of diester of 3-hydroxy octanoic acid, Cle alcohol, and Ce fatty acid. The major ions are mle 482, M+; m/e 383, M+-CsHllCO; m/e 366, M+-CSHllCOOH; mle 259, M+-C16H31; m/e 241, Mf-C16H&; m/e 159, M+-CSHIICO-CI.SH~~; mle 143, M+-C16H31-C5H~~COOH.

mle 143, 171, and 199 from the intact lipids was the same as that obtained by GLC/MS analysis of transesterified 3-hydroxy methyl esters done as indicated above. The SID was therefore used to calculate all values for the percentage composition of diesters in the uropygial glands of both injected male and female birds.

Quantitative Analysis of the Changes in the Uropygial Gland Lipid Composition Induced by Hormone Injection-The first noticeable change in the composition of the uropygial gland

Estradiol Induces Peroxisomes and Diesters in Mallards 9799

of females treated with estradiol and thyroxine was a decrease in the short chain content of the acyl portion of the wax esters (Fig. 4, bottom). By 8 days of treatment, the short chain ( S C ~ ~ ) acids that usually constitute 80-90% of the acyl portion had decreased to 35% and eventually decreased to 15%. Es- tradiol alone also caused a decrease in the content of the short chain acids but only to a much lower extent. Treatment of male mallards with estradiol and thyroxine also caused a dramatic decrease in the content of short chain acids that reached 10% in 12 days of treatment (Fig. 4, top). Without thryoxine, estradiol caused a slower decrease in short chain content that reached 35% in 16 days of treatment. These changes that occurred prior to the appearance of the more polar diester waxes were very similar to those observed during the postnuptial molt (eclipse) of mallards (4).

GLC/MS analyses of the uropygial gland lipids showed that the 3-hydroxy fatty acid diesters began to appear after the content of the short chain acids in the monoester waxes reached a minimum in the estradiol-treated females (Fig. 4). The diester content steadily increased until it became the only component. These changes were hastened by the addition of thyroxine with estradiol. For example, by 12 days of treatment with both hormones, the diester content reached nearly loo%, whereas it took 22 days of treatment with estradiol alone to reach this level of diesters. In either case, the composition of the diester waxes was identical to that observed with female mallards in the mating season (6). When the hormone treatment was discontinued, the diester waxes disappeared within a few days and the short chain monoester

J .- E?

2 6 10 14 18 22 26 30 2 6 10 14 18 22 26 30

Days

FIG. 4. The diester (left) and short chain fatty acid (right) content of the wax esters produced by the uropygial glands of male (top) and female (bottom) mallard ducks injected with 178-estradiol alone ( E only) or with thyroxine/l7j3-estradiol (T,/E). For T4/E and E alone, the injections were discontinued on days 14 and 20, respectively. The diester content is expressed as a percentage of the total esters, and short chain fatty acid content is expressed as a percentage of total fatty acids in the monoester fraction.

waxes reappeared as the dominant component of the uropygial gland lipid (Fig. 4).

Detailed analyses of the uropygial gland lipids of male mallards showed that they did not produce detectable levels of the diester waxes at any time during the year (4). However, treatment of the male mallards with estradiol resulted in the production of diesters just as in the case of female mallards (Fig. 4). Estradiol with thyroxine hastened the changes also in the males, and diesters became the only component produced by the uropygial glands by about 14-16 days of treatment with the hormone. GLC/MS analysis of the diesters showed that the chain length compositions of the acyl portion, the alcohol portion, and 3-hydroxy fatty acid portion of the diesters produced by the hormone-treated male mallards were identical to those found in the female mallards (data not shown). When hormone treatment of the males was discontinued the diesters disappeared and short chain monoesters became the dominant component in a few days (data not shown).

Changes in S-Acyl Fatty Acid Synthase Thioesterase Level in the Uropygial Gland Induced by Hormone Treatment-S- Acyl fatty acid synthase thioesterase gene expression was found to be suppressed during eclipse resulting in the decrease in short chain acid content (5). To test whether the hormone treatment caused a decrease in the level of the thioesterase that causes the release of short chain acids from fatty acid synthase, the high speed supernatant from the gland extract was subjected to sodium dodecyl sulfate-gel electrophoresis. The thioesterase protein band at the 30-kDa region showed much less intensity in the case of the hormone-treated animals (data not shown). To measure the thioesterase level, an immunoblot analysis (18) was done with antibodies prepared against the thioesterase (Table 11). This measurement showed that the gland from the hormone-treated male and female ducks contained only 20 and 16%, respectively, of the thioesterase found in the gland of the untreated counterpart. This level of thioesterase was identical to that found in the glands during the postnuptial molt called eclipse (Table 11).

Induction of Peroxisomes by Estradiol-Biochemical studies suggest that the 3-hydroxy fatty acid synthesis involved peroxisomal fatty acyl-CoA oxidase (8). To test whether peroxisomes are involved in the biosynthesis of the diesters, subcellular fractions from the uropygial glands of females that produce diesters during the mating season were examined for enzyme activities after sucrose density gradient centrifugation. NADPH-cytochrome-c reductase activity remained in the upper fractions, while catalase sedimented to a higher density region (Fig. 5A). Cytochrome-c oxidase assays suggest that the peroxisomal fraction was contaminated by some mitochondria. Electron microscopic examination showed diaminobenzidine-staining particles and mitochondria in the cat-

TABLE I1 Effect of treatment of mallards with thyroxine and estradiol on the level of S-acyl fatty acid synthase thioesterase i n urooveial elands.

S-acyl fatty-acid synthase thioesterase % Of

pg/mg soluble protein Untreated male 13.5 100 a Male treated with thyroxine/ 2.7 20

Male in eclipse 2.3 17 Untreated female 12.2 100 a Female treated with thyroxine/ 1.9 16

estradiol

estradiol a Males and females were hormone-treated for 24 and 14 days,

respectively.

9800 Estradiol Induces Peroxisomes and Diesters in Mallards

A

FIG. 5. A , distribution of enzyme activities following sucrose density gradient centrifugation of the 105,000 X g pellet from the homogenate of the uropygial gland of females producing diester waxes. @-Hydroxylation, 3-hydroxydo- decanoic acid formation from dodecanoyl-CoA (panel I ) ; diester formation, formation of diester from labeled 3-hydroxydecanoic acid (panel 3 ) ; esterification, wax ester formation from labeled hexadecanol (panel 4 ) . Fatty alcohol and 3-hydroxy fatty acid esterifying activities were associated with the ER and peroxisomal fractions, respectively. B, distribution of the enzyme activities following Nycodenz density gradient centrifugation of the 1000 X g supernatant from the homogenate of the uropygial gland of diester-producing mallards. Centrifugation and enzyme assays were done as described in the text. Diester formation (panel 4 ) was measured with hexadecanol and [ 1-"Cldodecanoic acid as substrates; only the labeled diester generated was considered the product. For @-hydroxylation (panel 5 ) total 3- hydroxy acid was measured. The amount of diesters found in the fractions by mass spectrometry are shown in arbitrary units of intensity of a selected diagnostic ion at mle 143 in panel 3.

4 n l 0 2

"", "" L""

6 1 2 1 8

FRACTION NO.

alase-containing fractions, whereas the ER fraction contained vesicles that did not stain with diaminobenzidine. For the present purpose, it was important to resolve the peroxisomal activities and the enzyme activities normally associated with endoplasmic reticulum. In fact, fatty acyl-CoA.fatty-alcohol transacylase that generates monoester waxes was found exclusively in the endoplasmic reticulum fraction, whereas fatty acyl-CoA oxidase involved in the synthesis of the 3-hydroxy fatty acid was located in the peroxisome fraction. Since the diester of the 3-hydroxy fatty acid is the unique component of the uropygial glands, catalysis of the esterification of the 3-hydroxy fatty acid to generate the diester would be a novel activity induced by hormone treatment. To test for such an activity, that has not been previously demonstrated in any cell-free preparations, the density gradient fractions from the gland were incubated with 3-hydro~y[2-~H]decanoic acid, ATP and CoA to activate the carboxyl group, and lauroyl- CoA and hexadecanol to esterify the hydroxyl group and the carboxyl group, respectively, of the hydroxy acid. Thin layer chromatographic analysis of the products generated by the particulate fraction, presumed to contain peroxisomes, revealed the formation of the diester. With such an assay, the esterifying activity that generates the diester from 3-hydroxy

B

FRACTION NUMBER

fatty acid was found to be located in the fatty acyl-CoA oxidase-containing peroxisomal fraction (Fig. 5A) . Acyl-CoA reductase, the enzyme that generates fatty alcohol for the synthesis of monoester waxes and for esterification of the 3- hydroxy fatty acids, was found both in the endoplasmic reticulum and in the peroxisome fractions (Fig. 5A) . Similar results were obtained with the uropygial glands of male mallards induced to generate diesters by hormone treatment (data not shown). These results suggest that endoplasmic reticulum is involved in the monoester wax synthesis and peroxisomes are involved in the diester wax synthesis.

To further define the subcellular location of the enzymes that catalyze the synthesis of the 3-hydroxy acid diester, Nycodenz gradient centrifugation was used to separate peroxisomes from mitochondria and ER. A catalase-containing fraction sedimented into the gradient, whereas mitochondrial cytochrome oxidase activity and NADPH:cytochrome-c reductase activity (ER) remained on top as expected (Fig. 5B) . Formation of 3-hydroxy fatty acid and diester formation were catalyzed by the catalase-containing fraction (Fig. 5B) . When the amounts of diester present in the Nycodenz gradient fractions were measured by GLC/MS, it was found that the fraction with the maximal amount of diester-forming enzyme

Estradiol Induces Peroxisomes and Diesters in Mallards 980 1

activities also contained the highest amount of diesters (Fig. 519, panel 3 ) . Electron microscopic examination of the catalase-containing fraction after diaminobenzidine-staining revealed that the fract,ion with the maximal amount of catalase activity contained particles of relatively uniform size that showed dark staining with very few other types of bodies (Fig. 6A) . The slightly lighter fraction that showed high catalase activity and maximal diester synthesizing activity contained intensely diaminobenzidine-staining bodies. In some of these microbodies small nonstaining regions were found that indicate accumulation of lipids (the diesters would not be expected to be stained). These isolated peroxisomes appeared similar to the diaminobenzidine-staining peroxisomes in the diester- producing gland tissue, as indicated below. Such microbodies were not found in glands that did not produce diesters. The lighter peroxisomal fraction also contained some mitochondria-like appearing bodies that were smaller in size than the normal mitochondria. The next lighter fractions contained the smaller mitochondria-like particles, large vesicles with double membrane (presumably of mitochondrial origin), and some peroxisome-like staining bodies. This fraction had low diester-forming activity. Since these fractions did not contain cytochrome oxidase activity, the nature of these small mitochondria-like bodies remains unknown. The cytochrome oxidase activity representing normal mitochondria was found at the top of the gradient as found previously (12). T o test whether the diaminobenzidine staining observed in the particles contained in the diester-forming fractions is due to catalase, staining was done in the presence of CN to inhibit cytochrome oxidase or aminotriazole to inhibit catalase. The staining of the microbodies was abolished by aminotriazole

A

FIG. fi. A , electron micrographs of diaminohenzidine-stained par- t irks from catalase-containing fractions ohtained hv Nvcodenz gradient centrift~gation shown in Fig. 5H. The particles ohtained from individunl fractions indicated hy the numhers were examined and representative views are shown. Examination of the different regions of the pellet showed the following general picture. Frncfion 19showed relatively uniform size diaminohenzidine-staining particles with verv k w other tvpes of particles: /roction 21 showed diaminohenzidine- staining peroxisome-like microhotlies, a few small mitochondria-like I)odies and a few other small vesicles. Frncfion 27 showed small tnitochondria-like hodies, large vesicles with douhle memhranes. some smaller vesicles, antl a few tliaminohenzidine-staining peroxisome- like microhodies. The scnk. hnr indicates 0.5 pm in all panels. No lead stnining was used. /i, electron micrographs of the microhodies contained in the pooled diester-forming f'ractions from a Nycodenz gradient centrifugation similar t o that shown in Fig. 513. Top, tliami- rlol~c~nzidine-st;~itlingas done in A ; rniddlr, diamino1)enzidine staining in t h r presence o f 2 mM <'N; hottorn, diamind)enzidine staining i n t h c , presence o f 'LO mM aminotriazole (45) . No poststaining was used.

but not by cyanide (Fig. 6 H ) . Therefore, it is concluded that the diester-forming fractions contained peroxisomes.

If the peroxisomes are, in fact, involved in the diester production, the uropvgial glands of the females in the mating season and the glands of the males or females induced t o generate diesters by hormone treatment should reveal the presence of peroxisomes. To test this possihilitv, the uropygial glands were perfused with gluteraldehyde and treated with diaminobenzidine to stain peroxisomes. Electron microscopic examination showed that the uropygial glands of both the male and female mallarcls normallv contained a large number of oil-filled bodies similar to those described before in other avian uropygial glands (1, 11, 23); the usual subcellular stnlc- tures and some microperoxisomes associated with the endoplasmic reticulum, but very few peroxisomes could be detected. On the other hand, the uropygial glands from the females in the mating season showed a large number of diaminobenzidine-staining peroxisomes (Fig. 7 ) . The glands from both estradiol-treated female birds in nonmating season and males treated with estradiol/thyroxine showed a large number of diaminobenzidine-staining structures t.ypical of peroxisomes. The ultrastructural appearance of the uropygial glands of females in the mating season was indistinguishable from that of the hormone-treated animals (Fig. 7 ) .

DISCLSSION

Sebaceous glands have been thought to he under hormonal control (24-29). However, molecular level studies on the regulation of sebaceous gland metabolism is lacking at least in part because there are no convenient model systems. Our finding that the composition of the uropygial gland secretion changes dramatically during the mating season in the female and during the postnuptial molt in both male antl female mallards (4-6) suggests that this may be a good model for biochemical and molecular biological studies on the hormonal regulation of sebaceous glands. However, such studies would be extremely restricted if the experiments have to be limited to short periods during which the birds naturally undergo the changes. Therefore, we sought a way to induce changes that are similar to the natural changes during other times of the year. Treatment with estradiol induces compositional changes in the uropygial glands that mimic what happens in the natural cycle. Just as in the natural cycle, estradiol-induced changes also cause replacement of the short chain monoesters with long chain monoesters followed by the appearance of the diesters. In both cases, in the course of the natural cycle or at cessation of hormone treatment, the diesters are replaced first with long chain monoesters, followed by the appearance o f short chain monoesters. The molecular events involved would have to be elucidated before determining, in rletail, how the changes in the natural cycle compare with those induced by the hormone.

The hormonal changes which occur in mallard ducks during and immediately following the mating season have heen previously investigated. Much attention was focused on steroid and thyroxine levels in the natural life cycle ( 3 0 - 3 2 ) . 1)rlring the breeding season, estrogen levels were found to be n t the i r peak in female mallards ( 3 3 ) . We discovered that this is also the time when the 3-hvdroxv fattv acid diesters are naturally produced as the sole products of the uropygial gland ( 6 ) . Estrogen levels are also at their highest levels in males rlrlring the eclipse (X{ ) , the time when S-acyl fatty acid synthase thioesterase gene expression is suppressed ( 5 ) . Therelnre, we postulated that estrogens might be involved in causing the dramatic changes in the composition of the uropygial gland secretion during and after the mating season. In fact. inject inn

9802 Estradiol Induces Peroxisomes and Diesters in Mallards

FIG. 7. Electron micrographs of diaminobenzidine-treated mallard duck uropygial glands. 7'fJp /c / t . control male injected with olive oil; hottorn / r / f , male injected with thyroxine and 17B-estradiol ('/',/E) suspended in olive oil; right, female in nonhreeding state; hottorn right, female in breeding state. Both the T,/E-treated male and the hreeding female show large numbers of peroxisomes. The diaminohenzidine- stained peroxisomes appear as uni/orrn/.v dnrk hodirs (arrows point to groups of peroxisomes). The scnlr hnrs indicate 0.5 um.

male: mntrol lemale: non-breedm,:

of estradiol caused suppression of the thioesterase gene expression as suggested by the drastic lowering of the level of the thioesterase caused by the hormone treatment. I 9' mce thyroxine is known to augment the effects of steroid hormones in some other systems (34,35) and because elevated thyroxine levels have been detected during the natural cycle, we injected thyroxine with estradiol. In both the male and the female birds, thyroxine hastened the changes caused by estradiol but thyroxine alone was unable to cause detectable changes in the composition of t h e uropygial gland secretion. The mechanism by which thyroxine augments estradiol action in this system is not understood. It is not known whether thyroxine caused an increase in the number of estradiol receptors in the uropygial gland as recently suggested in the case of pituitary tumors (36).

During the natural eclipse, the level of the thioesterase enzyme and mRNA detected by hybridization with cloned thioesterase cDNA and by in vitro translation was found to be drastically reduced (5). In the present study, the enzyme level was found to be similarly reduced by estradiol injection. However, the mechanism by which estradiol causes suppression of the thioesterase gene expression is not known. The thioesterase gene from the mallard that has been cloned and sequenced contains six introns (37). The first intron, but not t h e 5"flanking region of this gene, contains consensus se- quences typical of estrogen receptor binding sites. Whether a transacting factor actually binds this region of the gene and suppresses the expression of this gene is not known.

The diester synthesizing activity is associated with the peroxisome-like microbodies. A combination of the results obtained from the chemical, biochemical, and electron microscopic examination supports this conclusion. The results obtained with the fractions from the sucrose density gradient centrifugation showed that the diester synthesis is associated with peroxisome fraction contaminated with mitochondria but not in the endoplasmic reticulum that catalyzes wax ester synthesis. The Nycodenz density gradient fractionation clearly separated the normal mitochondria, containing cytochrome oxidase, from the peroxisomal fraction. The diester synthesizing activity was associated with the catalase-con-

! * . : 1 \ . ! 1 a , ! ~ r v , c : : : m L :

taining peroxisomal fraction. Electron microscopic examination of the individual catalase-containing fractions showed that the heaviest fraction (fraction IS), with the maximal level of catalase activity, contained fairly uniform size diaminohenzidine-staining microbodies with a t-ypical peroxisomal appearance. The slightls lighter fraction (fraction 21), containing catalase activitv and the maximal diester synthesizing activity, showed the presence of diaminohenzidine- staining microbodies, and some small mitochondria-like hodies. That these mit.ochondria-like bodies are probably not involved in diester svnthesis was indicated hy the finding that a yet lighter fraction (fraction 23) containing higher amounts of such small mitochondria-like bodies had much less diester synthesizing activity. The fractions containing diester synthesizing activity contained little cytochrome oxidase activity. whereas normal cytochrome oxidase-containing mitnchondria were found near the top of the gradient as expected (12). That the diaminohenzidine staining of the microbodies in the tlies- ter-forming fractions is most prohahlv due to the catalase activity was demonstrated by the inhibition of staining by aminotriazole but not by cyanide. The catalase-containing fractions that showed the enzymatic activities involved in diester synthesis also contained mass spectrometricallv measured diesters. The peak of catalase activity was in a fraction heavier than that which contained the penk of diester concentration and diester svnthesizing enzyme activities, although it is clear that the diester synthesizing fractions contained large amounts of catalase. The probable reason for this is that the diester accumulation would render such peroxisomes slightly lighter than the other peroxisomes. The conclusion that diester svnthesis involves the peroxisomes is supported not only by the location of the enzvmes in the peroxisomal fractions, but also bv the finding that onlv diester-producing glands showed the presence of significant numbers of peroxisomes. In further support. of this conclusion is the fact that fatty acyl-CoA oxidase, previouslv shown to be involved in the synthesis of the diester (8), is well known to be a peroxisomal enzyme. However, the remote possihlitv that the diester synthesis occurs in a subclass of peroxisomes or a novel

Estradiol Induces Peroxisomes and Diesters in Mallards 9803

class of microbodies that are difficult to separate from peroxisomes cannot be ruled out.

Hormone induction of peroxisome proliferation in the target tissue for the production of sex pheromone during a specific period to enhance reproduction gives a new role for peroxisomes. Hypolipidemic drugs, environmental contami- nants such as phthalates, and even high fat diets are known to induce peroxisome proliferation in the liver (38, 39). Per- oxisomes were once thought to be mainly a catabolic organelle that plays a major role in P-oxidation of fatty acids. In recent years, many biosynthetic functions have been found to be associated with peroxisomes (40-44). The subcellular location of fatty acyl-CoA oxidase involved in the synthesis of the 3- hydroxy fatty acids and esterification of the 3-hydroxy fatty acids involved in the formation of the diesters in the peroxisomal fraction strongly suggest that the diesters are synthesized in the peroxisomes. Even the acyl-CoA reductase that would generate the fatty alcohol needed for the esterification of the hydroxy acid was found in the peroxisomal fractions, supporting the hypothesis that the peroxisomes contain all of the necessary enzymes to convert fatty acids to diesters of 3- hydroxy fatty acids, a sex pheromone of the duck.

Syntheses of monoester waxes and diester waxes require fatty acids and fatty alcohols. Thus, the metabolic switch from monoester waxes completely to diester waxes is intriguing in that monoester waxes are not made, even though the acid and alcohol components required are produced during the period when diester waxes are the sole products of the uropygial gland. The subcellular location of the enzymic activities involved in the synthesis of the two types of waxes reported in the present study suggests that the switch from monoesters to diesters is achieved by compartmentalization. Thus, to achieve the production of diesters, a compartment that specializes in the synthesis of the diesters, peroxisomes, is produced during the mating season. Proliferation of peroxisomes in the uropygial gland of the duck, specifically during the mating season when the diesters are naturally produced and when induced to produce the diesters by hormone treatment, is consistent with the postulated novel role for peroxisomes. The biochemical reason for the complete disappearance of monoesters is not clear. It is possible that one or more of the enzymes involved in monoester formation becomes limiting, or the proliferation of peroxisomes might result in consumption of much of the substrates for diester formation.

Hormonal induction of peroxisome proliferation in the target tissue is a novel observation. Injection of the hormone into the breast muscle caused peroxisome proliferation in the uropygial gland but there was no indication of peroxisome proliferation in the liver. Injection of clofibrate, a hypolipidemic drug known to induce peroxisome proliferation in the liver, did not induce peroxisome proliferation in the uropygial gland (data not shown). Thus, the hormonal induction of peroxisome proliferation appears to be specific to the sebaceous gland. Other target tissues of steroid hormones were not examined. It is tempting to speculate that a prolonged period of intake of estrogens could cause proliferation of peroxisomes in the breast tissue and contribute to breast cancer as previously suggested for the induction of liver cancer by peroxisome proliferators (46). The mechanism by which steroid hormone induces the synthesis of peroxisomes is not known. It is intriguing that what appear to be microperoxisomes are present in close association with intercellular membrane stacks in the glands that produce monoester waxes (1, 11, 23, 40), but the larger peroxisomes of the type that are usually observed under conditions of peroxisome proliferation

appear in significant numbers only in the diester-producing glands. In the absence of a satisfactory method to isolate the microperoxisomes, biochemical comparison between the two types of peroxisomes is difficult. I t is not known whether incorporation of hormone-induced enzymes, such as fatty acid oxidase and esterifying enzymes that are involved in diester synthesis, into the microperoxisomes can lead to the formation of the peroxisomes observed in the diester-producing glands. In any case, with the uropygial gland system, it is possible to examine the nature of new gene expression patterns induced in this gland by the hormone treatment and thus get clues as to how steroid hormones trigger peroxisome proliferation.

Acknowledgments-We thank Kathy Wolken for her assistance in electron microscopy, Dale Adkins for technical assistance, and Drs. Richard Burry and John M. Robinson for assistance with interpre- tation of the electron micrographs.

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estradiol induces proliferation of peroxisome-like microbodies and

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