[31 and

BIOLOGY

REPRODUCTION

OF

In Vitro

43,

913-921

Differentiation Luteal-like

(1990)

of Bovine Theca Cells: Morphological MEIDAN,2

RINA

GIRSH,

ELI

Department

ORLY

of Animal

Hebrew

The

and Granulosa and Functional BLUM,

Science,

and

EDITH

Facu4y

The

of Jerusale,n,

University

Cells into Small Characteristics1

and

Large

ABERDAM

of Agriculture

Rehovot

Israel

76100,

ABSTRACT This

study

luteal-like insulin-like

cells

was

cells.

imum

ninth

characteristics and

pattern

intensified

in luteinized

cholera

toxin

theca

(100

progesterone

ng/ml),

results

the for

the

cells

cell

types

also

indicated

resulting

On

proceeded

in a 4-fold

were

whereas

9, cells

were

to cAMP

steroidogenesis

corpus

luteum

that

regulation

elevating

under

of several

tains two distinct luteal vious studies indicated UI and hCG by small cells

respond

trast, terone

large cells, than do

erately

to very

basal

of steroidogenesis

agents.

lutea dients yield

to physiological which small

high

for secretion

LGC,

on

in LGC and

the

other

mammalian

species

July

Received

October

11,

was

presented

Development

‘Correspondance: hovot

76100,

elevated

theca a max-

functional

progesterone lipid

droplets

9 days

in culture,

progesterone

levels

production

ng/ml),

same

and

and

After

into

Lg/ml),

reached

Numerous

cells.

in progesterone

by LTC; the

unable

forskolin

treatments

doses

to be different:

to be

of UI

[1-3].

Similarly,

cAMP

stimulated

cell

[31 and stimulation

from studies bovine and ovine

preparations. A different

was (10

ob-

&M),

failed

to stimulate

highly

dependent

or

LTC are but

nonetheless

are

able

to

International Faculty

Tiberias,

cytometry

in this of small

combining

has

improves approach

the

been

purity

to obtain

described (95%)

gra-

[3];

of large

separated

study. It is based and large luteal

density

luteal

this luteal

cells

on the observation cells, at least until

was that mid-

cies [9-14] to luteinize granulosa and theca cells into large and small luteal-like cells, respectively. Luteinization was assessed by use of several morphological and functional criteria.

their

MATERIALS

diverse [6].

AND

METHODS

Materials

that emcorpora

Dulbecco’s Modified Eagle Medium/Ham’s Nutrient Mixture F12 (1: i; DMEM/Mix F12); was obtained from Grand Island Biological Co. (Grand Island, NY); antibiotics and fetal calf

Symposium

serum

(IGF-I) many).

Re-

Israel.

913

(FCS)

(Kibutz

Beth

brand, (24 Denmark).

Chemical

Israel.

of Agriculture,

methodology

cycle, was shown to be the theca and granulosa layers, respectively, of the Graafian follicle [81. This study was designed to test the ability of factors known to induce progesterone biosynthesis in follicular cells of several spe-

analogs

Nunc trup, 1989,

flow

greatly

undertaken the origin

1989.

in part at the Ares Serono and the Ovulatory Response, It Meidan, Dept. of Animal Science,

a new

and

method

In con-

luteal cells include of the phospholipid-de-

Recently

dients

con-

9, 1990.

Accepted

Follicular

and

stimulated

cells.

LH (10

LTC appears

are

80%).

of UI [3,4].

was obtained dissociated

hand,

tries

on

and

(2

granulosa

morphological

5 days;

3 h with

in vitro

insulin

in culture

of luteal

drop

luteinized

saM),

of culture,

basal

an additional

challenged

secrete much higher basal progescells, appear to respond only mod-

doses

information enzymatically

work

days

in luteinized

a dramatic

from which luteal cells were separated by density gra[7] and elutriation centrifugation [4]. These methods luteal cells with variable degrees of homogeneity (60-

‘This

day

3-5

be

(10

conditions.

calcium-activated protein kinase of calcium mobilization after UI

This ployed

to those

for

first

diameter,

steroidogenesis

medium

cell types, small and large cells. Premarked differences in response to and large luteal cells in vitro. Small

bovine small and large responses to activators

pendent profiles

comparable

could

exhibited

insulin

cell

in contrast

or activators of the adenylate cyclase system were also found to be more active in ovine and bovine small luteal cells. The inhibitory effect of prostaglandin F2a (PGF2a) previously described in nonseparated luteal cells [5] is exerted on large luteal cells only [3]. Other functional differences between different

the after

that

cells

of forskolin

only plus

found

in progesterone

increase

interna

During

rose

active

INTRODUCTION The

presence

of forskolin

in basal

(LGC), Day

theca

the

agents.

It was

both

cells

(LTC).

luteum.

staining culture

granulosa

corpus

and in

progesterone

in the presence

incubated

replication

9 days

of these

respectively;

from

and

for

or a combination

adrenodoxin

demonstrate

responsive

sustain

Cells

granulosa

bovine

cultured

in LGC.

These and

of cell

by luteinized

served

were

ng/ml),

isolated

withdrawn,

maintained

on

cells

mitochondrial were

stimulants

whether

cells

androstenedione,

of culture.

of luteal

secretion,

were

day

theca

1(100

and

estradiol

on the

to investigate and

factor

growth

secreted

and

undertaken

Granulosa

Co. was The

(St.

were

obtained

Haemek,

Israel).

wells/plate, Biochemicals Louis,

16-mm were

MO).

obtained from kit to determine

from Tissue

Biological culture

diam.; obtained

Insulin-like

Boehringer-Mannheim estradiol was

Indus-

plates

were

Nunc, Kampsfrom Sigma Growth

Factor (W.

obtained

Gerfrom

I

914

MEIDAN

Pharmatred

Co.

3H]Progesterone,

(Petach

Tikva,

3H]estradiol-17[3 search Products

were (Boston,

purchased MA).

of Hormone Rehovot, Israel).

odoxin,

purified

ously mone

[1,2,6,7-

from

provided Research,

and

from

Antisera against androstenedione, estradiol were generously provided partment Science,

Israel).

[1 ,2,6,7-3H]androstenedione,

bovine

NEN

Weizmann raised

adrenal

by Dr. I. Hanukoglu Weizmann Institute

1713(De-

Institute of against adren-

cortex,

were

(Department of Science).

at least

mm,

Re-

progesterone, and by Dr. F. Kohen

Research, Antibodies

washed

[2,4,6,7-

DuPont

ET AL.

gener-

of HorAntiserum

and

3 times

by centrifugation

resuspended

FCS. The cells Approximately

in culture

were 7-9

then X 10

at 200

medium

X g for

containing

5 1%

counted and tested for viability. theca cells were recovered from

each follicle. Granulosa cells (0.5_i x 105/well) and theca cells (2 x 104/well) were cultured in culture medium plus 1% FCS only or in culture medium plus 1% FCS in the presence of forskolin, insulin (2 .tg/ml), IGF-I (100 ng/ ml), forskolin media were

plus changed

insulin, or forskolin plus IGF-I. Culture and the cells retreated every 48 h over

against oxytocin was kindly provided by Dr. A.P.F. Zoological Society of London, Institute of Zoology,

Flint (The London,

a 9-day period. After 9 days of culture, cells were incubated in medium containing only 1% FCS for an additional 5 days (9 + 5); culture media were replaced after two days (9 +

U.K).

Hormone

2). At each

Bovine

Pituitary

LH was

Program

obtained

from

(Baltimore,

the

National

MD).

trypsin, Isolation

and mm

and

of Granulosa

Experimental Ovaries after

Theca

Cells,

Cell

counter.

at a local containing

but without contralateral

abattoir a large

less than 20 follicle (1.2-

Short-term

an active corpus luteum in the ovary were chosen. Aliquots of

follicular fluid were collected from these follicles for rapid determination of their estradiol content, using an ‘251-E2 kit. Estradiol-active follicles whose follicular health status corresponded to the criteria established by McNatty et at. [151 were with

used in this study. a rubber policeman

were MIX

flushed F12),

several

The follicles were gently to remove the granulosa

times

containing

14

with g/L

culture

sodium

scraped cells and

medium and heparin

100 0.25 and

U/

.tg/ml 10 g/

ml DNAse. It has been reported that luteal cells cultured in the presence of fungizone do not respond to LH [16]. Yet in another study, bovine luteal cells were cultured up to 210 h in fungizone-containing on progesterone response fungizone was present cultures in the presence and could not detect of the

cells.

200 X g for 5 mm, medium containing with a hemocytometer blue

dye

exclusion.

to

media without dibutyryl-cAMP

in long-term or absence any difference Granulosa

From

a typical

were

follicle,

with

forceps

and

incubated

for

theca from

EDTA)

and

were

kept

for

further

For each correspondby trypsinization (0.05%

counted

by

centrifuged

was then chopped, 45 mm in culture

57

of Luteinized

Stimulation

a Coulter

particle

Cells

Granulosa and theca cells treated sulin for 9 days were used for these

with forskolin experiments.

plus inOn Day

9, cells were washed several times with medium (without antibiotics or serum) and then challenged with bLH (10 ng/ ml), cholera toxin (CT; 100 ng/ml), or forskolin (10 p.M) for 3 h. At the end collected for steroid ized

and

of the incubation determination,

and

period, cells

media were were trypsin-

counted.

X

lO

in 0.2 5%

at

viable

Production

Spent culture media obtained at 1, 3, 5, 7, 9, 9 + 2, and 9 + 5 days of culture were assayed for 17 13-estradiol, androstenedione, and progesterone by RIA, using specific antisera stenedione,

[19, 20]. The sensitivity and progesterone

6.4%,

respectively. and interassay

12.5%,

and

androstenedione Determination

respectively, 5.2%

of CXtytocin

and

11.8%

for progesterone for

and interassay respectively,

cells. 4.5%, and

1713-estradiol.

Secretion

Spent culture media of granulosa and theca assayed for oxytocin content by RIA as described rick and Flint [21]. Values are expressed per intra10%,

androand 7.8

expressed per io of variability were

Values are coefficients

10.5%, and

limits of estradiol, assays were 3.9, 7.8,

cells were by Sheldcells. The

coefficients of variability were 2% and and the sensitivity limit was 10 pg/ml.

tryp-

any remaining et al. [18]). The

and the fragments medium containing

of Steroid

Determination

pg/tube, The intra-

interna layer was the theca externa

15 mm

EDTA in PBS at 37#{176}C to dissociate cells (as described by Bendell

theca interna incubated for

effect Since

cultures, we carried out of this antimicotic agent in the steroidogenic cells

cells were obtained. The by gently peeling it away

fine

an [17].

decanted, and resuspended in culture 1% FCS. The cells were then counted and assessed for viability by ttypan

granulosa removed sin/0.02% granulosa

media

(DMEM/

bicarbonate,

ml penicillin, 100 p.g/ml streptomycin, fungizone supplemented with 50 U/mI

response

0.02%

Cultures,

spent

oxytocmn determination. cells were detached

Design

were collected slaughter. Ovaries

1.5-cm-diam.) ipsilateral or

replacement,

steroid and ing culture,

were 3 mg/

ml collagenase and 10 pg/ml DNAse. The tissue fragments were then passed several times through a Pasteur pipette,

Immunofluorescence Granulosa mm diam.) plates and staining Goldring

Microscopy

and theca cells were plated on circular (12glass coverslips placed in 24-multiwell culture treated as detailed above. Immunofluorescence

for adrenodoxin et at. [22].

was

performed

as described

by

IN VITRO

Determination Cell

of Cell

diameter

Statistical

OF

recorded

before

period after equipped

plating

and

trypsinization by use with a micrometer.

at the of an

Analysis

Steroid

concentrations

cell diameter the General

were Linear

ysis System (SAS tiple Comparisons nificant derived iments

in culture

media,

cell

number,

analyzed by analysis of variance, Models procedure of the Statistical Institute test.

Inc., Cary, Differences

NC) and Tukey’s were considered

when p < 0.05. Each data point from several individual follicles as indicated in the figure legends.

and using AnalMulsig-

Dayl

(mean ± SEM) is in separate exper-

Estradiol

Production

Estradiol the

of the tured ture,

by Granulosa

content UI

of the

surge

follicular

and

bovine

declines

cells

in vitro exhibited cells supplemented

Cells preovulatory sharply

begins

[23, 24].

follicle

rises

when

lutemnization

Granulosa

cells

3 of culture,

a similar pattern: with exogenous

io

cells

days

and

estradiol was

sharply

negligible

after 24 h in cultestosterone (300

decreased

to about

in all treatment

Thecal

tissues

preovulatory tion

by Theca

Production

in bovine

are

follicles. follicles

the

site

to estradiol,

decreases

FO

#{149} INS

groups

when

Androstenedione production stimulated by forskolin and

bined

insulin

with

forskolin

5

in the

androgen

secre-

lutemnization

or IGF-I

EJ

INS.

c:

FO

#{149} OF

is mi-

still