96

Biochimica

et Biophysics

Acta,

531 Biomedical

0 Elsevier/North-Holland

BBA

(1978) Press

96-108

57255

SYNTHESIS OF LIPOPROTEIN GRANULOSA CELLS

PATSY

M. BRANNON,

Division

of Nutritional

(Received

April

5th,

ANNE

LIPASE IN CULTURED

H. CHEUNG

Sciences,

and

ANDRk

Cornell University,

AVIAN

BENSADOUN

*

Ithaca, N. Y. 14853 (U.S.A.)

1978)

Summary Avian granulosa cells cultured as a homogeneous parenchymal population contain lipolytic activity. This activity is stimulated 2-5-fold by serum, inhibited 90% by 1 M NaCl and inhibited 80% by specific anti-lipoprotein lipase immunoglobulins. 85% of the activity binds to heparin-Sepharose 4B, and 70% of bound activity is eluted with 1.5 M NaCl. Thus, the lipolytic activity of cultured granulosa cells is lipoprotein lipase. Granulosa cells were shown to synthesize lipoprotein lipase in culture by incorporating [3H]leucine into the enzyme protein, as measured with an immunoadsorption technique. Finally, colchicine was shown to increase intracellular lipolytic activity, suggesting an inhibition of secretion of this enzyme by cultured granulosa cells.

Introduction Lipoprotein lipase (diacylglycerol acyl-hydrolase, EC 3.1.1.34) is the sole enzyme responsible for the hydrolysis of chylomicron and very low density lipoprotein triglyceride [1,2]. There is considerable evidence that this hydrolytic step is necessary for the transfer of lipoprotein triglyceride fatty acids from plasma to extrahepatic tissues [l-3]. Indirect evidence suggests that the functional lipoprotein lipase is present on the luminal surface of capillary endothelial cells. The site of synthesis of lipoprotein lipase has not yet been clearly demonstrated. In order to establish the locus of production of this enzyme and investigate its regulation, several reports have appeared on the study of lipoprotein lipase in cultured heart cells [4,5], adipose tissue stromal cells [6] and cultured 3T3-LI fibroblasts [ 71. However, these cultured cells are heterogeneous populations of different cell types. This study presents the characterization of lipoprotein lipase in cultured avian ovarian granulosa cells. Because avian granulosa cells are present in the ovary as a single cell layer firmly * To

whom

requests

for

reprints

should

be addressed.

91

attached to the vitelline membrane, they could be easily separated as a homogeneous cell type. Further, the availability of specific anti-lipoprotein lipase immunoglobulins made it possible to demonstrate the incorporation of [ 3H]leucine into cellular lipoprotein lipase. Materials and Methods Materials. Diaminobenzoic acid, EDTA, triolein (Sigma, grade I), crystallized albumin, oleic acid, Triton X-100, and colchicine were obtained from Sigma Chemical Co., St. Louis, MO. Glycerol tri[l-14C]oleate was obtained from Dhom Products, N. Hollywood, Calif. Medium 199 (with Hank’s salts (10 times), chicken serum, 2.5% trypsin, 10 000 U/ml penicillin, 10 000 g/ml streptomycin, 25 g/ml Fungizone, 200 mM glutamine, 7.5% NaHCO,) was obtained from Gibco, Grand Island, N.Y. Sterile Falcon tissue culture flasks (75 cm2 growth area) were purchased from VWR Scientific Co., Rochester, N.Y. Gelman a-6 metrical filter (0.45 ~_lrn pore size) were obtained from Fisher, Rochester, N.Y. Sepharose 4B was purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. Cyanogen bromide was obtained from Eastman/VWR Scientific Co., Rochester, N.Y. [3H]Leucine was obtained from Dhom Products, N. Hollywood, Calif. (l-2 Ci/mmol) or from Amersham-Searle, Arlington Hts., Ill. (>lOO Ci/mmol). Phosphatidylcholine was isolated from egg yolk by a modification of Singleton’s procedure [S]. Unbleached heparin was obtained from Inolex, Chicago, Ill. Cell culture. Mature follicles from laying white leghorn hens (7--14 months old) were aseptically removed and maintained in sterile phosphate-buffered saline (0.15 M NaCl/lO mM phosphate, pH 7.35) at 37°C. Granulosa cell layers were isolated from the follicles as described previously [9] and washed extensively with sterile phosphate-buffered saline at 37°C to remove traces of yolk. Cell layers were collected and maintained in sterile phosphate buffered saline at 37°C. Then they were transferred to sterile tubes and sterile 0.25% trypsin/ 1 mM EDTA in phosphate-buffered saline (approx. 1 ml/3 follicles) was added. Cells were dissociated with the trypsin/EDTA for 2-3 min at 37°C. Sterile chicken serum (0.2 ml/3 follicles) or Media 199 with 15% chicken serum (1 ml/3 follicles) was added to stop the reaction. The cells were centrifuged at room temperature at a low speed (250 X g) for 2 min. After removal of the supernatant, the cells were resuspended in Media 199 (1X) containing 15% chicken serum (heat inactivated at 56°C for 30 min), 50 U/ml penicillin, 50 pg/ml streptomycin, 5 pg/ml Fungizone, and 0.9% Na2C03. An aliquot of cell suspension was used for a viable cell count in Trypan blue with a hemocytometer. Cells were seeded in flasks (75 cm2 surface area) at a density of 3 or 3.5 . 10’ cells and incubated with 7 ml medium. The cells were incubated at 37°C in a 5% CO, atmosphere. Medium was changed after the first 24 h and every 48 h thereafter. Cell harvesting. At designated times, cells were harvested by treatment with 3 ml 0.02% EDTA in a buffered salt solution (290 mosM per 1: 8 g NaCl, 0.2 g KCl, 1.15 g Na,HPO,, 0.2 g KH,PO, and 0.2 g glucose) for 5 min. Dislodged cells were collected into a tube and centrifuged for 5 min at 1000 X g at 15°C. Cells were resuspended in 0.5 ml phosphate-buffered saline and an aliquot

98

taken for DNA determination. Acetone/diethyl ether powders were made of the cells after the addition of 100 p g bovine serum albumin 191. Lipase activity was extracted from the powder with 100 ~1 0.7 M NaCl~40% glycerol/5 mM sodium Verona1 buffer (pH 7.0) for 15 min on ice, following a 10-s sonication with a Biosonik IV low speed probe. The extracts were then centrifuged at 4’C at 1000 Xg for 5 min. Aliquots of the extract were used for protein analysis [ 101 and for determination of lipolytic activity. Analyses. DNA was determined by a microfluorimetric filter analysis [ 111. Lipase activity was determined using a synthetic ~14C]triolein substrate emulsified with phosphatidylcholine (phosphatidylcholine:triolein ratio of 0.065) in 0.15 M NaCl. In a total volume of 0.5 ml, the assay system contained 2.5 mM triolein, 0.1 M NaCl, 5 mM CaClz, 0.2 M Tris, 1% crystallized and lyophillized albumin and 30 p 1 rat serum (heat-inactivated for 30 min at 56°C) [ 121. A 60-~1 aliquot of extract of the acetone powder of the cells was assayed for 60 min at 30°C. The reaction was stopped by the addition of 1.9 ml CHCl,/ CH30H/C6H, (2 : 2.4 : 1, v/v; with 0.18 mM oleic acid). Free fatty acids were separated by the addition of 62 ~1 1 M NaOH [13]. Following a 15-min centrifugation at 15°C and 1000 X g, 1 ml of the upper phase was taken for scintillation counting in 8 ml Aquasol (New England Nuclear, Boston, Mass.). Lipase activity is expressed as nanoequiv~ents (nequiv.) of free fatty acid released per h. Light and electron microscopy studies. To establish the homogeneity of the freshly isolated cells, granulosa cell layers were fixed in Bouin’s solution, sectioned transversally and stained with hematoxylin eosin for light microscopic examination. Freshly isolated cells and cells in culture for 4 days were compared by electron microscopy. Isolated granulosa cell layers were washed in phosphate-buffered saline and fixed in 2% glutaraldehyde in phosphate-buffered saline and post-fixed in 1% 0~0,. After dehydration in a series of graded concentrations of acetone, the cells were embedded in Epon Araldite. Cells in culture were washed in the flasks and fixed similarly. Dehydration was done in the flasks in a series of graded concentrations of ethanol and cells were embedded in Epon [14] _ Thin sections were obtained in a Sorval MT-2B ultramicrotome and stained in 2% uranyl acetate and lead citrate. Sections were viewed in a Philips EM 300. Antibody inhibition of lipolytic actiuity. Antiserum to purified avian lipoprotein lipase was prepared in the goat or in the rabbit, as described previously [ 11. Partially purified anti-lipoprotein lipase immunoglobulins were isolated using Na2S0, precipitation and DEAE-cellulose ion-exchange chromato~aphy 1151. To examine the effect of anti-lipoprotein lipase serum on granulosa cell lipolytic activity, flasks of confluent cells were harvested and pooled. Aliquots of extract of acetone powder prepared from cultured granulosa cells were incubated with varied amounts of purified immunoglobulins of normal rabbit serum or anti-lipoprotein lipase serum (with titer of 26.0 nequiv. fatty acid/h per I-(g immunoglobulin in a total volume of 0.2 ml containing 20% glycerol/O.2 M NaCl/lO mM phosphate buffer (pH 7.4)). Following incubation at 21°C for 60 min, the mixtures were centrifuged at 20 000 X g in a Beckman 40.3 rotor for 30 min. 200 ~1 of each mixture were assayed for lipase activity (Fig. 6A).

99

A second procedure was also used, in which partially purified immunoglobulins were incubated with aliquots of extracted acetone powders of pooled granulosa cells. In this incubation, total immunoglobulin protein was constant in each assay tube, but the proportion of control immunoglobulin and antilipoprotein lipase immunoglobulin (with a titer of 9.7 nequiv. fatty acid/h per p g immunoglobulin) was varied. Incubation mixtures containing 20% glycerol/ 0.2 M NaC1/0.2% albumin in a total volume of 0.2 ml were reacted at 21°C for 60 min. 200 ~1 of each mixture were assayed for lipolytic activity (Fig. 6B). Heparin-Sepharose 4B affinity chromatography. Heparin was coupled to activated Sepharose 4B [ 161 and a 2.5 X 1.0 cm column prepared. The column was equilibrated with 0.15 M NaC1/30% glycerol/l0 mM phosphate buffer (pH 7.0), and an extracted sample of acetone powder of pooled granulosa cells was applied in the same buffer. Following a 25-30-ml wash with 0.5 M NaC1/30% glycerol/l0 mM phosphate (pH 7.0), the enzyme activity was eluted with 1.5 M NaC1/40% glycerol/l0 mM phosphate (pH 7.0)/3% albumin. Incorporation of [3H]leucine into lipoprotein lipase. The [3H]leucine incorporated by granulosa cells into lipoprotein lipase was determined by immunoadsorption. Cells were exposed to [ 3H]leucine (5 p C/ml media) for 1, 2, 3 and 6 h. 20 ml goat antiserum (titer of 13 058 pequiv. fatty acid/h per ml) was coupled to 125 ml activated Sepharose 4B by the method of Neuwelt et al. [ 171. At each sampling, cells from three flasks were harvested and pooled. Acetone powder was prepared and extracted with 200 ~1 of the extracting buffer defined above. Each incubation mixture contained 120 ~1 extract and 200 ~1 50% suspension of immunoadsorbent in a total volume of 520 1_r1containing 0.15 M NaC1/20% glycerol. Each mixture was reacted at 4°C for 3 h, then washed sequentially with 2 ml quantities of 20% glycerol, 2 M NaC1/20% glycerol, 0.5 M NaC1/0.2% Triton/BO% glycerol and 0.15 M NaC1/20% glycerol. All previous solutions were prepared in 10 mM phosphate buffer (pH 7.0). The immunoadsorbent gel was then counted in 8 ml Aquasol to determine 3Hlabeled protein radioactivity. Lipolytic activity was determined at 1, 2, 3 and 6 h and corrected for 3H-labeled protein radioactivity with duplicate assays containing non-radioactive triolein substrate. In some experiments, the 3H-labeled protein bound to immunoadsorbent was dissociated with SDS and subsequently digested at 100°C with 1% mercaptoethanol for 1 min. Aliquots of the digests were run on 10% polyacrylamideSDS slab gel electrophoresis [ 181. Results The cell layer obtained after washing consisted of a single layer of granulosa cells, attached firmly to the vitelline membrane on the apical portion of the cells (Figs. 1 and 2). The more loosely associated basement membrane is visible in the light micrographs (Fig. 1A). Electron micrographs of the fresh cells (Fig. 1B) exhibit ultrastructural features similar to those described for mature preovulatory follicles from hens [ 191 and pigs [ 201. Cultured granulosa cells have ultrastructural features essentially similar to those of fresh cells with ample electron dense particles (Fig. 2). Granular endoplasmic reticulum is, however, much more prevalent in cultured cells than in the original cell layer.

Fig. 1. Original granulosa cell layers. CA) Parafin section stained with hematoxrlin 625X). (B) Electron micrograph (magnification 17 500X; 1 I.rm= 17.5 mm).

and eosin (magnifica-

tion

Following seeding, cells from 2-4 flasks were harvested at intervals and DNA was determined as a measure of growth. The results of 3 experiments are summarized in Fig. 3. Plating efficiency (percent initial DNA attaching in 24 h)

101

Fig. 2. Electron

micrograph

of cultured

4-day-old

granulosa

cells (:magnification

17 500x;

1 pm = 17.5

mm).

was 20% in most experiments. DNA was relatively constant in the first 72 h, then increased 3-fold to a maximum by 120 h. At 120 h, cells observed microscopically formed a confluent cellular layer. Cells were polygonal and contain many intracellular vesicles, of which a large percentage stain with Oil Red 0. These results suggest that the cells exhibit a period of rapid growth from 72-120 h. DNA decreased and cells were found clumped together and also detached from the surface of the flasks after 120 h.

Characterization of granulosa lipdytic activity Flasks of confluent granulosa cells were harvested and pooled. Afiquots of the extracted acetone powder were assayed under one of three conditions: control, no serum in assay, or 1 M NaCl in assay. The lipase activity of granuloss cells was stimulated 2--5-fold by serum and inhibited 90% by 1 M NaCl (Table I). The inhibition of granulosa lipolytic activity by increasing concentrations of purified anti-lipoprotein lipase rabbit immunoglobulins is presented in Fig.

102 S.O-

z g 4.0= "E 0 3.0? \ g 2.0a

z 0

I.0

01

1 0

24 48 72

96 120 144 168

Time Fig.

3.

area. At

Growth

Each

eyery

of

point

cultured (0)

sampling

(hr)

granulosa

represents

time

the

in each

cells

seeded

mean

of

experiment.

at a density

4 experiments 2 flasks

of

3.0-3.5.

(sampled

wcrc

10s

O-144

measurf~d.

cells

per

75

cm*

h) or 3 experiments

Bars reprcsciit

surface (lF8

the standard

error

h). of

the mean.

4A. Maximum inhibition (82%) occurred at concentrations of 10 p g and greater. Similarly, maximum inhibition (80%) occurred where 8 pg or more purified anti-enzyme goat immunoglobulins were used (Fig. 4B). Lipolytic activity was also found in granulosa cells passed with trypsin/ EDTA dissociation for five generations. 8 confluent flasks of the fifth passage were harvested and pooled. Aliquots of the extracted acetone powder were assayed in triplicate with serum, without serum, or with 1 M NaCl in the assay. Activities were 84.3 + 7.9 nequiv./h per mg protein for control, 47.5 i 5.3 nequiv./h per mg protein for assay without serum, and 16.9 + 2.6 nequiv./h per mg protein with 1 M NaCl in assay. Thus, activity in passed cells is also stimulated by serum and inhibited by high salt.

TABLE

I

CHARACTERIZATION

OF

LIPOLYTIC Lipolytic

ACTIVITY

activity

Experiment Complete --

system

Serum

+ 1 M NaCl * Figures pooled **

Figures pooled

represent

IN CULTURED

(nequiv./h

1 *

55.2

+ 5.0

230.8

+ 2.0

120.4

3.9

? 1.0

37.3

f

S.E.M.

means

of

triplicate

CELLS

per mg protein)

Experiment

11.2

GRANULOSA

2 **

+ 23.0 r

4.0

? 12.0

analyses

for

each

extract

of

acetone

powder

of

12

of

12

flasks. represent flasks.

means

t

S.E.M.

of

quadruplicate

analyses

for

each

extract

of acetone

powder

103

160 A

160 0

I ' Anti-

I1

2 LPL

/

,111

I

4 6 6 IO 12 " 100 Immunoglobulin tug /tube)

Fig. 4. (A) Inhibition of granulosa cells lipolytic activity by rabbit anti-adipose lipoprotein lipase immunoglobulin. 14 flasks of confluent cultured granulosa cells were harvested and pooled on day 4 of culture. Acetone/ether powder was prepared and extracted (see Materials and Methods). Aliquots of extract were incubated with normal rabbit immunoglobulin (0) or rabbit anti-lipoprotein lipase immunoglobulin (m) (titer of 26 nequiv. free fatty acids/h per Hg immunoglobulin). Lipolytic activity in aliquots incubated with normal rabbit serum was 60.2 nequiv. fatty acid/h per mg extracted protein (100% control). (B) Inhibition of granulosa cells lipolytic activity by goat anti-avian adipose lipoprotein lipase immunoglobulins. Confluent flasks were harvested and pooled on day 4 of culture. Aliquots of subsequently extracted acetone powders were incubated with a total of 12 c(g (0) or 100 pg (m) of total immunoglobulin protein with varied proportions of normal or anti-lipoprotein lipase immunoglobulin (titer of 9.7 nequiv. fatty acid/h per pg immunoglobulin) as indicated. 100% of control activities are 19.3 and 35.7 nequiv, fatty acid/h per mg protein for extract incubated with 12 and 100 pg immunoglobulin, respectively.

The use of heparin-Sepharose 4B for affinity chromatographic purification of lipoprotein lipase has been well established for avian adipose lipoprotein lipase [ 11, pig adipose lipoprotein lipase [ 121, as well as milk lipase [21] and post-heparin plasma lipolytic enzyme [22]. As a further means of identifying the lipolytic activity in granulosa cells, the binding of lipolytic activity of acetone/diethyl ether powder extracts to heparin-Sepharose 4B was determined. Typically, 85% applied lipolytic activity (400.6 nequiv./h) was bound to the column. A representative elution profile of lipolytic activity with 1.5 M NaC1/40% glycerol/3% albumin/l0 mM phosphate (pH 7.0) is presented in Fig. 5. A single peak of lipolytic activity eluted under these conditions, accounting for 70% of the lipolytic activity bound to the column.

104

The lipolytic activity of cultured granulosa cells appears to be lipoprotein lipase, as it exhibits the classical properties of the enzyme: serum stimulation, high salt inhibition, inhibition by specific immunoglobulins to highly purified avian adipose lipoprotein lipase, and elution from heparin-Sepharose 4B at 1.5 M NaCl. Lipolytic activity in cultured granulosa cells Cells were prepared as described in Methods. Duplicate flasks were sampled every 24 h and lipoprotein lipase activity, DNA and protein determined in 3 experiments (Fig. 6). Total lipoprotein lipase activity increased from 4 pequiv. free fatty acid released/h per flask at 24 h to 13 nequiv./h per flask at 120 h after seeding. Lipoprotein lipase activity per unit DNA remained stable through

180

2 \

140-

3

120-

‘, .-

IOO-

g

80-

(Cl l_PLlrnQ PROTEIN

2

Fraction

(0.4ml./tube)

0 24 48 72 96 120144168 Time

(hr)

Fig. 5. Elution of lipolytic activity of granulosa cells from a heparin-Sepharosr 4B column. 32 confluent flasks of granulosa cells were harvested and pooled on day 4 of culture. 3 ml of extracts of acetone powder (containing 133.5 nequiv. fatty acid/h per ml) was applied to a 2.5 X 1 cm heparin-Sepharose 4B column equilibrated with 0.15 M NaC1/30% glycerol/l0 mM phosphate (PH 7.0). 85% of applied activity bound to the column. Enryme activity eluted with 1.5 M NaC1/3% albumin/30% glycerol/l0 mM phosphate (PH 7.0) and 0.4-ml fractions were collected. 85% of applied activity was recovered. Fig. 6. Lipoprotein lipase activity in cultured avian graaulosa cells. Total lipoprotein lipase activity (A), activity/unit DNA (B) and activity/unit extracted protein (C) of cultured granulosa cells were determined. Each point represents the mean of three experiments. In each experiment, at every sampling time, two flasks were employed for individual determinations. Bars represent the standard error of the mean.

105

168 h after plating; however, there was a broad range of variation. activity per unit protein was stable from 24-120 h after plating, decreased.

Lipolytic but then

[3HlLeucine incorporation Incorporation was linear from 2-6 h (Fig. 7). During this period, lipoprotein lipase activity/mg protein was relatively stable (2.0-3.7 nequiv. free fatty acid released/h per mg protein). 10 flasks of granulosa cells exposed to [3H]leucine (14.0 Ci/ml media) for 36 h were harvested and pooled. The resultant acetone powder was extracted with 600 ~1 buffer. 230 ~1 extract was incubated with 766 ~1 50% suspension of immunoadsorbent. The radioactive profile of the slab gel of the 3H-labeled protein dissociated from immunoadsorbent is presented in Fig. 8. A single peak of 3H-label was observed. In a stained replicate sample containing 5 pg purified adipose lipoprotein lipase as carrier, a single stained band, which migrated with the same mobility as purified adipose lipoprotein lipase, was observed. Effect of colchicine on lipolytic activity in granulosa cells At 72 h after plating, flasks were divided into groups of 6 each. Each group was incubated with no colchicine, 2.5 . lo-’ colchicine, 2.5 . 10e6 colchicine, or 2.5 . lo-’ colchicine in the growth medium for 25 h at 37’C. Following incubation, lipolytic activity, DNA and protein were determined. Total cellular lipolytic activity per flask, activity/pg DNA and activity/mg protein increased (Table II).

I2

3

Time

4

5

6

(hrs)

Fig. 7. Incorporation of [3HIleucine into lipoprotein lipase. On day 4 of culture. cultured granulosa cells were exposed to 5 PC/ml media [3H]leucine. At each sampling, three flasks were harvested and pooled. 200 ~1 extract of acetone powders was incubated with 200 ~1 50% suspension of anti-avian lipoprotein lipase immunoadsorbent. Lipoprotein lipase activity was 2.0-3.7 nequiv. fatty acid/h per mg protein in this experiment.

106

I

2

3

45670 cm.

Fig. 8. Radioactive profile of slab gel electrophoresis of 3H-labeled protein bound to anti-avian lipoprotein lipase immunoadsorbent. 10 flasks of cultured granulosa cells exposed to 14 fiC/ml media [3Hlleutine from day 3-5 of culture were harvested and pooled. An aliquot of extracted acetone powder was incubated with anti-lipoprotein lipase immunoadsorbent gel. Bound 3H-labeled protein was dissociated from the immunoadsorbent with 150 ~1 3% SDS/10 mM phosphate (PH 7.0) at room temperature for 15 min. The mixture was spun at 2000 X g to remove immunoadsorbent. Dissociated protein was digested a 1OO’C for 1 min with 1% a-mercaptoethanol. 56 fil digested protein was electrophoresed on SDS-lo% polyacrylamide slab gel. It was then cut into 0.7 cm segments that were digested overnight in 0.2 ml 30% Hz02 at 50°C [251. Digests were counted in Aquasol. The RF values for adipose lipoprotein lipase and the 3H band of protein dissociated from immunoadsorbent were 0.221 and 0.219. respectively.

TABLE

II

EFFECT CELLS

OF CHOLCHICINE

TREATMENT

ON LIPOPROTEIN

LIPASE

ACTIVITY

Cells were grown in culture for 72 h, then incubated with treatments indicated harvested and analyzed. Figures represent means * S.E.M of 6 observations. Treatment

Average

nequiv./h

2.5 2.5 2.5.

1O-5 M M 1O-7 M

. 10-b

6.26 12.12 21.72 19.33

for 25 h. Cells were then

LPL activity

Total activity/flask

Control Colchichine-treated:

OF GRANULOSA

LPL activityfug

DNA

LPL activity/mg

%

nequiv./h

%

+ 2.56

100

1.81 ? 0.9

100

52.2

r 0.78 * c 3.2 * _+3.9 *

194 347 309

4.79 9.08 5.90

210 398 259

78.6 114.2 100.4

* Values significantly different from respective of variance and linear contrast methods [261.

control

? 0.6 * + 1.7 * * 1.4 *

nequiv./h

values at P < 0.01 as analyzed

protein %

? 15.3

100

+ 3.9 * + 11.4 * i 13.4 *

150 219 192

by one-way

analysis

107

Discussion The major advantage of using avian granulosa cells in studies of the regulation of lipoprotein lipase is that they can easily be isolated and cultured as a single parenchymal cell type. Careful dissection of avian follicles leads to the separation of intact sheets of single-cell-thick granulosa cells attached to a basement membrane and the vitelline membrane. Since lipoprotein lipase is thought to be synthesized in parenchymal cells and subsequently transported to endothelial cells [ 21, the presence of variable ratios of both cell types in cultures would complicate studies on the regulation of this enzyme. Published reports both on heart cells and on adipose tissue stromal cells employed cell populations that contain an unknown proportion of nonparenchymal cell types [ 6,241. Cultured avian granulosa cells grow in reproducible pattern after initial seeding of flasks at 3.35 * 10’ cells per flask. Growth of cells as measured by the DNA content of the flask is low in the first 72 h and is very rapid in the 72-120-h interval. It has been shown previously that freshly dissected avian granulosa cells have lipoprotein lipase activity [9]. This activity has a specific activity of 1.62 pequiv. of fatty acid released/h per mg protein, about half that of avian adipose tissue. Cultured granulosa cells also have lipoprotein lipase activity that is present throughout the culture period and in passed granulosa cells. The properties of the lipase of cultured granulosa cells (stimulation 2-5-fold by serum, 90% inhibition by 1 M NaCl, 80% inhibition by specific anti-lipoprotein lipase immunoglobulins and characteristic chromatographic behavior on heparinSepharose 4B) identified this lipolytic activity as lipoprotein lipase. Following trypsin/EDTA dissociative treatment, the specific activity of lipoprotein lipase of granulosa cells is much lower (0.21 pequiv. fatty acid/h per mg protein) than that of intact, isolated cells not treated with proteolytic enzyme. Lipoprotein lipase activity in cultured cells declines even lower, to 0.06 pequiv. fatty acid/h per mg protein, by 24 h. This further decline in specific activity of the enzyme in cultured cells may represent the absence in the growth medium of one or more of the factors that regulate the synthesis, degradation, activity and secretion of lipoprotein lipase. Nonetheless, total lipoprotein lipase activity in cultured granulosa cells increased from 4.8 to 14 nequiv./h per flask in the period 24-120 h after culturing. During this same period, the specific activity of the enzyme (lipoprotein lipase activity per unit DNA or lipoprotein lipase activity per unit protein) was relatively stable. This suggests that lipoprotein lipase activity may be closely associated with the number of cells present, as cell growth is maximal 72-120 h after culturing. Granulosa cells in culture have been shown to synthesize lipoprotein lipase, as observed in the incorporation of [3H]leucine into the enzyme. The validity of the immunoadsorbent technique as a means of isolating labeled lipoprotein lipase from extracted acetone powders was assessed by SDS slab gel electrophoresis of labeled protein bound to immunoadsorbent. The observation of a single peak in the radioactivity profile of gel and of a single staining band of protein that migrated similarly to purified lipoprotein lipase suggests that only lipoprotein lipase binds to the immunoadsorbent.

108

Colchicine is known to interfere with microtubular function. Stein et al. [23] have shown that colchicine treatment of perfused rat heart leads to a marked decrease in hep~n-releasable lipoprotein lipase. Also, colchicine treatment of the whole rat decreases heparin-releasable plasma lipoprotein lipase [ 241. Since microtubules may function in intracellular transport of secretory vesicles, colchicine impairment of microtubular function is likely to inhibit transport processes. In this study, colchicine treatment increased lipoprotein lipase activity in cultured granulosa cells. Colchicine increased total lipoprotein lipase activity and activity per unit DNA 2-3-fold in 24 h, indicating that cultured granulosa cells may secrete lipoprotein lipase activity. Acknowledgment This work was supported by the United nal Institutes of Health grant HL 14990.

States Public Health Service, Natio-

References 1

Kompiang,

2

SCOW,

3

Schotz,

P.,

K.O.,

Bensadoun, Twu,

M.C.,

Biochim.

Biophys.

4

Chajek,

5

Henson,

6

Rothblat.

7

Eckel,

A.

and

Blanchette-Mac&e,

T..

Acta

Stein,

L.C.,

0.

489,

and

(1976)

Smith,

M.E.,

.I. Lipid

L.C.

Chen,

Res.

(1976)

C.H.,

17,

498-505

Circulation

Garfinkel,

Res.

A.S.

39,

and

149-162

Borenstajn,

J.B.

(1977)

214-224

Stein,

M.C.

and

DeMartinis,

Fujimoto,

M.

and

Pedersen,

Schotz.

G.H. R.M.,

J.S.,

Yang,

E.J.

Y.

and

(1977)

F.D.

W.Y.

Biochim.

Hararu,

and

Biophys.

I. (1977)

(1977)

Biochim.

Biochem.

Brunzell,

J.D.

Acta

488,

Biophys.

Biophys.

(1977)

140-144

Acta

Res.

487.

212-221

Biochem.

Commun. Biophys.

78, Res.

45-50 Commun.

78,

288-

293 8

Singleton,

9

Benson,

W.S.. J.D.,

10

Bensadoun,

11

Cattolico,

12

Bensadoun,

13

Khoo,

14

Luft, Levy,

16

March,

17

Neuwelt,

18

Swaney,

19

Wynbum,

20

B&sing,

MS..

Brown, A.

and

A. and Weinstein, R.A.

and

A.,

J.C.,

15

Gray,

Bensadoun,

J.H.

Gibbs,

Steinberg, (1961)

H.B.

and

S.C.,

J. Biophys. Sober,

Parikh,

E.A., J.B.

Frank, and

G.M.,

Johnston, Z.

and

LEVY, (1976)

H.S.

P.R.

(1976)

J. Biol.

Med.

103,250-252

Exp.

Biol. Anal.

(1976)

Biochem.

J. Biol. Biophys.

Aitkan,

T. (1972)

(1974)

J. Biol.

R.N.C. Anat.

(1966)

446,

Z.

Zellforsch.

Microsk.

Chajek,

T..

Stein,

0.

and

Stein.

Y.

(1975)

Biochim.

Biophys.

Acta

388,

260-267

24

Chajek,

T.,

Stein,

0.

and

Stein,

Y.

(1975)

Biochim.

Biophys.

Acta

380,

127-131

25

Roheim,

and

Cochran,

Chem.

(1972)

D.

249.

2220-2227

2882-2890

and

Anat.

72,

5345

82.173-186

23

Edelstein,

251,

5752-5758

Walter.

H.,

53-56

561-565

H.,

Hirsch,

J. Biol.

Acta

251,

Egelrud,

V.W.

42.

60.149-152

Chem.

Greten,

Brown.

Chem.

Chem.

21

B. and

Sot.

148.347-350

572-582

22

P.S.,

Olivecrona,

Oil Chem. Med.

70.241-250 69,

V.W.

P. (1974)

Microsk.

Biol.

9.409-414

Sot.

Biochim.

and

Zellforsch.

J. Amer. Exp.

Brown.

Vagelos.

C.C.

(1965) Sot.

Biochem. and

Cytol.

Proc.

Cuatrecasas.

K.S.

I,. (1967) T. and

(1960)

J.J. and

Kuehl,

J.J.

J.L. Proc.

Biochem.

D.

Bioehem.

H.A. I. and

Anal.

Steinberg,

Huang,

White,

Anal.

(1975)

C.,

D.,

and

D. (1975)

D. (1976) S.P.

Ehnholm,

M.L.

Cohen,

247,

FEBS

Lett.

6212-6217 27,

Rachmilewitz,

306-310

D.

(1972)

Biochim.

Biophys,

Acta

278,

517-529 26

Snedecor. Ames,

Iowa

G.W.

W.G.

(1967)

Statistical

Methods,

6th

edn.,

Iowa

State

University

Press,