Cholesteryl esterase and endogenous cholesteryl ester pools in ovaries from maturing and superovulated immature rats

501

Bicnzhimicart Biophyaiae AC& 618 (1980) 501-509 @ Bhwier/North~HaiIand

Biomedical Press

BBA 57682

CIIOLFSTERYL ESTERASE AND ENDOGENOUS CIIOLESTERYL ESTER POOLS IN OVA&IES FROM MATURING AND SUPEROVULATED IMMATURE RATS ROBERT CHARLES TUCKEY and PATRICIA MARGARET

Department of Biochemietry, 6009 (Auetralia)

STEVENSON

University of Western Australia. Nedlande, Western Au&alla,

(Received October 8th, 1979)

Key words: Choleeteryl eetemse; Choleoteryl ester; Development; (Rat ovary)

Isotope dilution;

Results from the assay of cholesteryl e&erase (EC 3.1.1.13) with radiolabelled substrate are difficult to interpret if endogenous cholesteryl ester is present, We overcame this problem by using an isotope dilution method to measure the endogenous pool sizes of cholesteryl ester in subcellular fractions of the ovary. This permitted calculation of the total cholesteryl e&erase activity of the mitochondrial, microsomal, and cytosolic fractions of the ovary. At all stages of ovarian development most cholesteryl e&erase activity was found in the cytosol, and generally there was more activity in the microsomes than the mitochondria. The cholesteryl e&erase in all three fractions exhibited higher activity with cholesteryl oleate as substrate than with cholesteryl palmitate. Incxea~esin cholesteryl e&erase activity and endogenous ester concentration were found at two stages of ovarian development; firstly after initia tion of follicular growth by gonadotropin in the immature ovary, and secondly during luteinization. The increasee were observed in all three sub-cellular fkactions. Adminktration of human choriogonadotropin to rats which m lulxkized ovaries resulted in activation of the mitochondrial and microsomal chokteryl e&erase but not the cytosolic enzyme.

Cbolesteryl esters, located in lipid droplets in the cytoplasm of steroidogenic ti@ues, fun&ion as a store of chole&erol, the wrbstrate for steroid hormone @@r&a [1,2]. The esters are hydrolysed byeholesteryl e&erase which, in the ovary, is preeent in both the corpus luteum and interstitial tissue 131. Rehrman

502

and co-workers [4,5] have reported that the cytosolic fraction of the rat corpus luteum has the highest activity of cholesteryl e&erase and that the cytosolic enzyme is activated by lutropin. Bisgaier et al. [6] suggested that cholesteryl esterase in the ovary is activated by a similar mechanism to that in the adrenal cortex. In adrenal tissue cholesteryl e&erase is controlled by adrenocorticotropin via cyclic AMP and a protein kinase [ 71. Cholesteryl e&erase activity is usually assayed by measuring the hydrolysis of radiolabelled substrate. Most work to date has been done on the e&erase in the cytosolic fraction from which much of the endogenous ester can be removed by repeated centrifugation to remove lipid granules [ 1,4]. Because endogenous cholesteryl ester is present in the mitochondrial and microsomal fractions it is not possible to determine the total rate of ester hydrolysis with radiolabelled substrate in the usual way. We have used the isotope dilution method of Ichii et al. [S] to measure endogenous cholesteryl ester pools and total ester hydrolysis in mitochondrial, microsomal and cytosolic fractions of ovaries. The ovaries were from normally maturing rats aged 2042 days aM from immature rats superovulated by administration of pregnant mare serum gonadotropin and choriogonadotropin [ 91. Materials and Methods Chemical and mdiochemicals. Cholesteryl palmitate, cholesteryl oleate, pabnitic acid, cholesterol and aminophylline were obtained from Sigma Chemickrl Co., (St. Louis, MO, U.S.A.); cholesteryl [l-14C]palmitate and cholesteryl [l-“C]oleate from The Radiochemical Centre (Sydney, N.S.W., Australia); pregnant mare serum gonadotropin from Intervet (Artarmon, N.S.W., Australia) and human choriogonadotropin from Park, Davis and Co., (Sydney,.N.S.W., AustraIia). Radiolabelled cholesteryl esters were purified by thin-layer chromatography (TLC) on silica gel G with petroleum ether/diethyl ether/acetic acid (80 : 20 : 1, v/v/v) prior to use. Methanol was distilled before use. All other solvents and chemicals were A.R. grade. Tissues and subcellukrr fractionation. Ovaries were from Wistar albino rats aged 2042 days. 2Oday-old rats (designated day 0) were superovulated by subcutaneous injection of 60 I.U. pregnant mare serum gonadotropin and, 54 h later, 25 I.U. human choriogonadotropin. Groups of, 1540 treated or untreated rats were killed by cervical dislocation. Ovaries in each group were pooled, cleaned of oviducts and adipose tissue, weighed and homogenized in 5 vols. 0.25 M sucrose unless otherwise specified. Homogenates were centrifuged for 10 min at 600 Xg and the resuIti.ng supernatant centrifuged at 14 000 X g for 15 min. The 14 000 X g pellet was resuspended the original volume of 0.25 M sucrose and centrifuged at 8500 X g for 15 min. The 14 000 X g supernatant was centrifuged at 161000 X g for 40 min, the supernatant was removed from under the lipid granules, recentrifuged at 161000 X g for 40 min and the cytosol withdrawn from under the remaining lipid granules. The 14 090-161909 Xg pellet was resuspended in the original volume of 0.26 M sucrose and centri&ged at 161000 X g for 40 min to sediment the microsomes. All centrifugations were done at 24°C. Final mitochondrlaI and microsomal pellet were resuspended in 0.26 M sucrose at a dilu-

in

tion designed to give less than 50% ccmverkn of radhlabelled subs&ate b the ester&se assay. The cytosol wtbss-y diluted. T& 85oo--14 660 X gsuper, it cmw 7% of natant was not routinely assayed for ch the t&l activity in ovaries from day 6 wcperovufaaedrata, Measunentent of chotesteryt e&-mm a&My. 0.5 ml al the sukW hattion ws added to 0.5 ml of 0.2 M phosphate buffer, pH 7.4 for the cyto~~lic enzyme, pH 7.2 for the mitochondrial enzyme and pH 7.0 for microsomal e&erase. Tubes were preincubated for 3 min and the reaction was started by addition of the m~o~~~ed cholesteryl ester substrate. U~a~R~ cholesteryl oleate was added with the 14C-labelled ester to some tubes for calculation of the endogenous ester pool by isotope dilution. After incubation at 36’C, usually for 30 min, the reaction was stopped by adding 8 ml CHCl&H,OH (1 : 1, v/v) and the lipids were extracted by the method of Renston et al. [ 101. The dry lipid extract was applied as a band to 10 X 20 cm plates coated with 0.2 mm of Silica gel G, with 3 X 100 $ CHC&. Plates were developed in petroleum etherjdiethyl ether/acetic acid (80 : 20 : 1, v/v/v) and radioactive areas were located with a Varian Rerthold scanner. Lipids were also detected with Iz vapour and identified from reference compounds chromatogmphed simultaneously. Areas of silica gel corresponding to cbolesteryl ester and free fatty acid were removed and the 14C label counted in toluene eo~~g 4% CHJOH and 0.4% 2,5diphenyloxaxole. The remaining silica gel on ,the plate was also removed and counted and usually contained less than 2% of the recovered radioactivity. The silica gel ,did not affect counting efficiency. The hydrolysis of radiolabelled substrate was calculated from the radioactivity in the f&&y acid band expressed as a percentage of the total radioactivity recovered. Control values (boiled enzyme) were less than 1% and were substracted. Isotope ditu tion calculation. The isotope dilution method of Ichii et al. [S] as described by Coutts and Stansfield [ 111 was used to calculate endogenous ester pools and total hydrolysis of cholesteryl esters. The expression used is P = [Ca (Ar + AC) - CoArjfCo - Ca where P is tie endogenous pool (nmol), Cu is the % conversion in the presence of “C diluent, Co is the % conversion in the absence of 12C!diluent, Ar is the nmol of 14C substrate and AC is the nmol of ‘2C substrate {diluent). Equihbrium between substrate added and endogenous substrate was assumed. Pro&in determination. This was done by the biuret method, using bovine serum albumin as standard.

The cloister

f es term2 assay The rate of hydrolysis of cholesteryi oleate was not linear with incubation time. This non-linearity was probably due to the endogenous ester present. Rehrman and Armstrong [4] noted when working with the cytosolic &holesteryl esterase of luteinized rat ovaries that removal of endogenous cholesteryl ester was a prerequisite for obtaining direct pro~~on~~ between izxubation

TABLE I THE EFFECT OF VARYING THE AMOUNT OF VNLABELLED CHOLESTEROL DETERMINATION OF ENDOGENOVS ESTER BY ISOTOPE DILUTION

OLEATE ON THE

The experiment was done with the microsomaI fraction prepared from ovaries from day 6 superovulated rats. The incubation time was 20 min. Data for % hydrolysed 1s mean f SD. (n = 4). ’ 4C-labeIIed substrate

VnIabeIied + ’ 4C-IabelIed substrate

Endogenous ester

added (nmol)

% hydrolysed

added (nmol)

8 hydrolysed

nmol

11.48 11.48 26.48

21.11 f 1.38 21.17 i 1.38 16.40 f 1.42

26.48 46.48 46.48

16.40 f 1.42 10.88 i 2.06 10.88 k 2.06

11.4 11.9 12.9

time and enzyme activity. We found that the endogenous ester concentration calculated using the isotope dilution formula shown above was not altered by the incubation time used. The calculation of the endogenous ester concentration was also independent of the amount of unlabelled cholesteryl oleate added with the 14C-labelled substrate (Table I). Comparison of the rate of hydrolysis of “C-labelled substrate with the total rate of ester hydrolysis, for example in Table IV, illu8trates the importance of determining the endogenous ester pool size when measuring cholesteryl e&erase activity. pH optima of 7.2, 7.0 and 7.4 were determined for mitochondrial, microsomal and’ cytkolic fractions respectively. The mitochondrial fraction showed high e&erase activity at low pH indicating lysosomal contamination. A shoulder was present at pH 6.8 to 7.0 on the pH curve for the cytosolic enzyme suggesting that some microsomal e&erase might be present in the cytosol and that they are two distinct enzymes. Data presented previously ‘[ 121 indicated that ovarian cholesteryl e&erase was selective towards the cholesteryl fatty acid ester substrate, with preference for the CZZpolyunsaturated acids and that, of the two radiolabelled ester available to us, cholesteryl oleate might be a better substrate than cholesteryl palmitat& Table II, which shows the rates of hydrOly8eS of cholesteryl [l-14C]oleate and cholesteryl [ l-14C]palmitate by cholesteryl e&erase, confirms this observa-

TABLE II THE ACTIVITY OF CHOLESTERYL TERYL OLEATE A8 SUBSTRATES

ESTERASE WITH CHOLESTERYL

PALMITATE AND CHOLES-

Fractions were prepared from ovaries from day 6 superovulated rats. Tubes were incubated with 11.5 run01 of substrate. the cytosol for 10 min and microsomes and mitochondria for 20 min. Data are mean f S.D. (n = 3). Fraction

Activity (pmollmm per ma protein) Cbolerferyl [l-14C]pabnitatc

Mitochondria Microaomcs Cytosol

38* 6 211 f 13 111 f 13

hydrolysed

Cholesteryl [l- r4Cloleate hydrolysed 96* 1 434 * 23 277 * 24

505 TABLE III SUBCELLULAR MSTRIRUTION OF CHOLRSTRRYL AND S~RROVWLA~~ ~MATURE RATS

RSTRRASB

IN OVARIES

FROM YATVRMQ

Rata apd 20 days ware superovulated as described in MaterMs md kthods.

subosllular iraation

% dietrtbution of activity between eubcelhdu fraettone Time after initiatfon of superovulation (daya)

Age of maturation (days)

Mitochondria Microsomea cyt0s01

20

29

42

1

3

6

22.7 19.9 57.4

24.6 21.0

13.0 11.0 76.0

14.6 16.2 70.3

14.9 19.2 66.9

7.0 12.8 80.2

48.2

tion. In all fractions, the oleate ester was hydrolysed at least twice as fast as the palmitate ester. Throughout this paper cholesteryl oleate was used to measure e&erase activity.

The subcellular distributions of cholesteryl e&erase activity in ovaries from maturing rats and superovulated immature rats is shown in Table III. The distributions were calculated from the rate of hydrolysis of cholesteryl ester (determined by the isotope dilution method) and the amount of protein in each fraction. The highest proportion of the total cholesteryl e&erase activity was always in the cytosol. The d~~bution of cholesteryl e&erase in the rat ovary differs from the bovine corpus luteum where Gout& and Stansfield [ll’j, using the isotope dilution method, found the most activity in the mitochondrlal fraction and only a small proportion in the cytosol. It should be noted that in our data the 8500-14 000 X g supernatant fraction, which contains a mixture of ~~~~ particles, was not included so the total a&vi& associated with mitochondria and microsomes is slightly underestimated. Choiesteryl estemse activities during development of the ovary The cholesteryl e&erase activity in microsomal and cytosolic fractions of ovaries of untreated rats increased slightly in activity from days 20-29 after birth (Table IV). These rats were sexuaIly immature. By day 42 the rati had commenced their first estrous cycle and their ovaries contained several corpora lutea. Between days 29 and 42 when the corpora lutea developed there was a marked lncxe~ in the endogenous ester concentration and activity of cholesteryl e&erase, particularly in the mitocbondrial and cytxrsolk: fractions. The ovary of the 2O-day-old rat can be made to mature prematurely by the apron of pregnant ahare serum gonadotxopin and later human choriogonsrdotmpin. This treatment (superovulation) causes a large number of follicles to develop and luteinize simultieously [Q]. In the 24 h following the injection of pregnant mare serum gonadotropin there was an incre~ in the activity of cholesteryl e&erase in the mitochondrial, microsomal and cytotic fractions, the largest increase being 2.5fold for the cytosolic enzyme

606 TABLE IV CHOLESTERYL EZSTFJZASEACTIVITY AND ENDOGRNGUS CHOLESTERYL ESTER CONCENTRATIONS IN SUBCELLULAR FRACTIONS OF OVARIES FROM MATTING AND SUPBROVULATED IMMATURE RATS Rats aged 20 days were superovulated as described in Materials and Methods. Date for conversion of 14Clabelled substrate (3.3 nmol of cholesteryl [l- 14CIoleate) is mean f SD. (n). Unlabelled cholesteryl oleate was added as diluent (mitoahondria, 25 nmol; microsomes, 10 run01 and cvtosol 100 nmoll. in n determinations to calculate the endogenous ester concentration and total ester hydrolysis. Ester hydrolvsed is pmol/min per mg protein: endogenous ester concentration is nmol[g tissue. Mitochondria Ageof maturation (days)

14Clabelled substrate hydrolysed

20

4.8 f 0.1 (2) 3.7 f 0.4 (3) 9.5 f 0.5 (3)

29 42

Microsomes Endoaenous ester eoncn.

Total ester hydrolrsed

34

13

49

12

120

44

14Clabelled substrate hydrobsed 24.8 f 1.6 (2) 20.4 f 0.1 (2) 24.5 * 2.5 (4)

Time after initiation of ~pe~~ation (days) 1 4.8 f 0.2 136 26 24.8 f I.4 (31 (4) 3 43 f 0.1 103 23 38.6 f 0.1 (4) (4) 8 26.3 f 0.3 163 167 62.8 i 0.8 (3) (21

CYtosol Endogenous ester concn.

Total ester hydrolysed

14

42

38

57

67

14

37

67

50

118

246

612

14Clabelled substrate hydrolysed 8.6 * 0.1 (2) 8.9 i 0.5 (41 18.1 i 0.6 (41 6.8 i 0.5 (4) 8.2 f 0.4 (4) 40.8 t 0.9 (4)

Endoaenous ester concn.

Total ester hydrolysed

80

36

102

40

276

147

365

88

182

68

1011

796

(Table IV). There, Were larger increases in the cholesteryl e&erase activity between day8 3 and 6 of superovulation, during which time the follicles transformed into corpora lutea. This corresponds to the increases observed between days 29 and 42 of the maturing rat when corpora lutea from the fin& estrous cycle developed. The concentration of endogenous cholesteryl ester appears to be related to the activity of the e&erase, as increases in the activities were always accompanied by increases in the concentration of endogenous substrate avaiIable to the enzymes. ~tirn~~ti~n of cholesteryl e&erase activity by human chor~og~n~d~t~~in Acute stimulation of day 6 superovulated rats with human chorlogonadotropin, which increases the rate of steroidogenesis [ 133, resulted in the ,activation of the mitochondrial and microsomal cholesteryl e&erase, but the activity of the cytosolic enzyme was not increased (Table V). Activation of the mitochondrlal and microsomal enzymes by gon~o~op~ has not been reported previouzly to our knowledge. Behrman and coworkers f4,5] working with the superovulated rat ovary have shown s&nulation of the cytosolic cholesteryl e&erase in vivo by gonadotropin. Because our results disagreed with these authors we varhcd the conditions as follows in an attempt to show a&iv&ion of the cytosolic e&erase. In experiments 1 and 2 the conditions were those used

OF STIMULATION

OF DAY

6 SUPEROVULATED

RATS

WITH HUMAN

CHORIOGONADOTROPIN

ON CHOLESTERYL

ESTERASE

ACTIV-

Ratio -wd eonfrol

1.49

203 314

44

101

236

362

208

4

310

3

2

1

Mitoehondrie

414 773

1009 1490

711 840

1

3

4

Cytoeol

Microsomes

1190 1020

2

1065 1121

3

1121 370

4

and unlabeled cholesteryl oleete diluent sare wed to c&uTJw mans of four incuba~tionawith cholesteryI [l- i4CloIcate and four &ith chok.steryl I1 -‘4C]oleate lab the andogaurucl eetez concentratPon end total ester hydrolysis. The resulte of four experiments we shown; microwmal choleeteryl esteraee wee sot emeyed in experime?&~ 1 end 2. The ate were killed on day 6 of euperovulation. 30 ti after subcutaneous injection of 26 I.U. human choriogonadotropin in experiment 1. and 60 min leter in the other experiments. Results are rdven M pmol/min par mg pmtain.

THE EFFECT ITY

TABLE V

508

throughout this paper. In experiments 3 and 4 (Table V) the dose of human choriogonadotropin, given 54 h after the pregnant mare serum gonadotropin, was halved to ensure that most of the hormone had been cleared from the plasma by day 6 of superovulation when the effect of the acute dose of choriogonadotropin was studied. In experiments 3 and 4 the subcellular fractions were prepared and the e&erase assayed in buffer which in experiment 3 contained 0.25 M sucrose/O.1 mM phosphate/5 mM MgClJ2.5 mM aminophylline. Experiment 4 was the same except that MgCl? was excluded. The buffers used for fractionation in experiments 3 and 4 were pH 7.0 and 7.4 respectively. Cholesteryl e&erase activity in each fraction was assayed at its optimal pH. The cholesteryl e&erase in the cytosol was not activated under any of these conditions. Using methods already described Klinken [ 141, in these laboratories, showed that the cyclic AMP increased 6-fold while the rate of progesterone synthesis rose 60% when these superovulated rats were stimulated with choriogonadotropin. Discussion It has been reported that the cytosol can be prepared practically free of endogenous cholesteryl ester by repeated centrifugation to remove the lipid granules [1,4]. Using this procedure we were able to prepare cytosol from ovaries of immature rats with only about 0.8% of the total cholesteryl ester remaining. This is comparable to the 99% removal reported by Behrman and Armstrong [4]. Even with this efficiency of removal the endogenous ester concentration remaining was more than twice that of the “C-1abelled substrate added for the assay of cholesteryl e&erase. After the immature ovary had been stimulated to develop by gonadotropin, most of the cholesteryl ester remaining in the cytosol af& fractionation appeared to be free and not in lipid granules. This was reflected by a large increase in the endogenous cholesteryl ester concentration in the cytosolic fraction. It was 12.fold higher in the cytosol of rats 6 days after superovulation than in cytosol prepared from the ovaries of 20. day-old immature rats. We always removed lipid granules by the same procedure and unless there was a change in the nature of the granule it is unlikely that variation in the efficiency of their removal could account for the observed increase. Lipid granules from both interstitial tissue and corpora lutea appear to be membrane bound, have no cholesteryl e&erase activity and they can be used as substrate by the cyt&olic cholesteryl e&erase [5,15]. The release of free cholesteryl ester from the lipid droplets is probably necessary before ester hydrolysis by the cytosolic enzyme can occur. In the mitochondrial and microsomal fractions increases in e&erase activity were accompanied by increases in their endogenous ester pool sixes. The increases in endogenous ester concentration in these fractions may come about by the transfer of cholesteryl esters from the lipid granules to these organelles. The ovaries of 20dayold rats consist predominantly of interstitial tissue and immature follicles. The interstitial tissue is rich in cholesteryl esters which are stored in granules [16,17]. Following t&e injection of immature rats with pregnant mare serum gonadotropin, chol&eryl esters are mobilized [13,17]; the ovarian content of cholesteryl esters decreases while the amount of free cholesterol in the ovaries increases. This data, plus the increase in cholesteryl

e&erase activity at this,t&ne, ix@zate q&the mpbilizatim of~cholesteryl esters from the lipid granules in the interstitial tissue involves ester hydrolysis. Pregnant mare serum gon~o~op~ causes the cells of tbe follicles to divide and the ovary increases in weight from about 9 mg before treatment to about 80 mg 4 days later [14]. The hydrolysis of esters following gonadotropin stimulation of the immature ovary is probably to supply free cholesterol for membrane synthesis in the rapidly growing follicles. The conversion of cholesterol to steroid hormones at this time is very low [ 143. The admonition of human ~ho~ogonado~op~ to the rats 54 h after the first injection causes synchronous formation of corpora lutea in the ovaries. Luteinization begins 4 days after initiatioh of superovulation. In the corpus luteum the cholesterol released from the cholesteryl esters is used for intramitochondrial progesterone production [13,18]. Robinson 1191 has shown with isolated mitochon~a that hydrolysis of [ 4-14C]cholesteryl oleate occurs before the cholesterol is converted to [ 4-‘4C]progesterone. Stimulation of rats on day 6 of superovulation with human choriogonadotropin causes activation of the mitochondrial and microsomal cholesteryl e&erase but not the cytosolic enzyme. Behrman and coworkers [4,5] have shown activation of the cytosolic enzyme in vivo by lutropin. Their studies were done with luteal tissue from Ho&man rats at days 8-11 of superovulation. The unresponsiveness to gonadotropin of the chdesteryl esterase in the cytosol of the newly formed IuteJ tissue which we are using may be because there is a high basal rate of activity, it is more than twice as high as that reported by Armstrong and Flint [5 J. Cholesteryl esters are depleted from ovaries of day 6 superovulated rats following in vivo stimulation with human cho~ogon~o~op~ [ IZ]. Our results suggest that this depletion is brought about by activation of mitochondrial and microsomal cholesteryl e&erases. Acknowledgement This work was supported Grants Committee.

by grant 92 0975 from The Australian Research

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

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12,