BIOLOGY

OF

REPRODUCTION

46,

Oocytes

Promote

Mouse

1196-1204

BARBARA

(1992)

Proliferation of Granulosa Follicles In Vitro1

C. VANDERFIYDEN,3

The Jackson

EVELYN

Laboratory,

Cells from

E. TELFER,4

Bar

Harbor,

and

Preantral

J.

JOHN

Maine

and Antral

EPPIG2

04609

ABSTRACT Evidence on

the

is now

role

ferentiated

old

oocyte

mural

mice,

Cell

emerging

of the

and

granulosa

mural was

in granulosa

Qocytectomy

being

in unconditioned which

after

complexes

labeling

index

cell

effect

of FSH

one

or more

cumulus

cells

promoted

the

treated

more

OOX

with

FSH,

mural

a single

layer

of squamous

an unidentified a multilayered

ganized

as pseudostratified

to respond

to the oocyte-derived

at specific

stages

attached to the basement and cumulus granulosa

cally

coupled

ucts

have

to the

been

Februar

Received

November

‘This

research

of their

3Supported Canada. Road,

Ottawa,

‘Supported ent address: School

FAX:

[1, 21: mural

Differences

by NIH

J. Eppig,

(207)

address: Ontario,

granulosa

and

the folmetaboli-

in secretory

cumulus

Canada

of Ecology King’s

grant

HP

The Jackson

effect

600 Main

cells,

Street,

fellowship

from

of Medicine,

K1H

Bar

acid

Building,

University

Research of Ottawa,

Council 451

Resource

from

the Rockefeller

Management,

Edinburgh,

Scotland

Foundation.

University EH9

(23%

reduction

medium

had no effect

effect

indicate

that

factor(s);

in OOX

oocytes

the

No

secreted

of preantral

cells

terminally this

a negative

27%

medium.

granulosa

The

on intact

FSH had and

the

to bring

of conditioned

follicles.

differentiated indicates

observation

difference

that

in

disperses

the

or mucification expansion, but

cell of FSH

proliferation

their

response

to

cumulus

cells,

the stimhy-

a process

[3-5]. Mural granulosa become luteal cells.

tradiol

causes

studies,

induction

have

cells These

development,

presence

Pres-

nization

of Edinburgh,

of the

communication

3JG.

1196

and,

follicle, normally

on

escells

act synergistically in vivo. In numerand

disruption

provided

of es-

granulosa

function

hormones

of these studies of the three-dimensional

the

mitogenic

similarly,

by culturing

of various

factors; but the interpretation cated because of the loss

Smvth

cells

these two hormones cell proliferation examined

The

by its stimulation

granulosa of FSH-receptors

been

in the

[12-14].

mediated

by the

the growth,

cells

monolayers

of

in vivo

is probably

production

ulosa

8M5.

fellowship and

the Medical

in preantral

surge of gonadotropins. Gonadotropins granulosa cells to produce and secrete

tradiol

ous

23839. Laboratory,

however,

and

this

effect

cell and/or

FSH

culture;

samples

follicles.

a dramatic

granulosa

prod-

granulosa

period.

two cell types also differ in their distribution of receptors [6-8] and their steroidogenic capabilities (9-111. In experiments on intact and hypophysectomized animals, both estrogen and FSH have been found to stimulate

the or-

288-5079.

Department

by a post-doctoral

of Agriculture,

growth phase, subpopulations

intact

undifferentiated

called expansion do not undergo

to form the oo-

for an

development.

aluronic

to

22, 1991. John

Institute

In response

FSH

for 4 days,

reversed

complexes

(131, suggesting that to promote granulosa

by a post-doctoral

Present

oocyte’s into two

in mural

was supported

ME 04609.

cells,

of the

or without

follicles

preantral

proliferation

role

(OOX),

as monolayers

in intact

results

of antral

preovulatory ulate cumulus

folwith

14, 1992.

‘Correspondence: Harbor

primordial associated

membrane enclosing cells attached and

oocyte.

found

Accepted

only closely

epithelia

cells licle,

cells

including

contains oocyte

the

These

the

cultured

3-h

antral

with

stimulatory

the relatively

granulosa

only

granulosa cells proliferate of granulosa cells around

cyte. Toward the end of granulosa cells differentiate

cultures.

failed

cells

granulosa

signal, the epithelium

the

were

in monolayer

from

in intact

mice.

determine

with

dif-

12-day-

structure

an additional cells

of 32%

from

22-day-old To

cultured

medium

index

focuses more

last 24 h. Oocyte-cumulus

Oocyte-conditioned

prevented

by both

and

for

compared

a reduction

short-term

INTRODUCTION At birth the murine ovary licles that consist of a primary

samples.

were

complexes

index

isolated

cultured

for the

granulosa

cell

(2)

oocytectomized

fofficles

cells

group

study

and

three-dimensional

were

Preantral

added

of mural

labeling

FSH

proliferation

the

and follicles

Oocyte-conditioned

causing with

retaining

to each

labeling

of the

in these

cell

the

of intact

complexes,

cumulus

however,

in

follicles).

complexes

granulosa

were

in oocyte-cumulus

preantral

follicles

follicles

granulosa

This

gonadotropin-primed

preantral

5H-thymidine

index

were

and

of medium).

added

labeling

Both

in a doubling

differentiated

act on granulosa

can

the

a decrease

cell

of

was

follicles

index.

complexes

with

follicles

labeling

24 h. Mural

period,

to the level

resulted

on preantral

that

and

the factor

but

treatment

observed

factors

follicles

ovarian

The

was

in

in OOX

in oocyte-cumulus

complexes.

in

and follicles

complexes

for a further

groups.

from

preantral

5H-thymidine

while

oocyte

oocyte/l

5H-thymidine

resulted

complexes

on proliferation

then

an increase

reduction

(1

obtained

complexes

OOX

cells,

of granulosa

from

Preantral

of the

the and

for a 48-h

treatment

oocyte

removes

function cells

follicles.

were

cell

as intact

group

treated

antral

complexes

medium

to each

caused

71%

of OOX

that

as well

4 h and

among

of the and

oocyte-cumulus cumulus

for

found

removal

in OOX

effect

added

medium

were

the

follicles,

was

from

cells

and

granulosa

determination

or oocyte-conditioned

incubated

oocyte-conditioned

that

cells

of 24 h and then

were

granulosa

oocyte-cumulus

procedure

medium

no differences

proliferation,

granulosa

in the development

undifferentiated

oocyte-cumulus

Mural

period

complexes

and

a role

of (1)

by autoradiographic

cell

3H-thymidine

equilibration

cells

a microsurgical

or follicle.

plays

cumulus

and

quantified

of the oocyte

the oocyte proliferation

cells

granulosa

proliferation

complex

that the

in

of the by the

gap

of gran-

the

cells

and

as

growth

is compliorgaintercellular junctions

be-

OOCYFES

tween granulosa structure of these

cells, cells

several

studies

of these

and the [15-17].

forming

growth

factor (FGF) in combination pins,

factors-a [22,

regulate

23], with

and

cell

consideration

servations

that

the

oocyte

development and cocious luteinization death of the luteinization dition,

oocvte body

that

mouse

ulated mulus FSH

oocytes

oocyte

uble

secrete

Using

factors

that cell of gran-

in granulosa

a specific,

cell proliferation ganization of the

cumulus

cells

procedure

oocvtes

cuto

whereby

an oocyte-granulosa evidence suggesting promote

of granulosa

cells

the

cell comthat solgranulosa

in preantral

or00-

and

an-

tral follicles vitro models

is the subject of the present study. Three in were used to assess the potential effect of oo-

cyte-secreted

factors

lated from

on granulosa

cell

preantral follicles, 2) oocvte-cumulus antral follicles, and 3) mural granulosa

tral

follicles

in monolayer

Collection For

and

Culture

studies

using

AND

dispersion

following Waymouth

modification. MB 752/1

MO)

+ 3 mg/mI

Lisle,

IL)

+

transferrin,

Bedford,

ITS

and

cell complexes cells from an-

from using

previously

The ovaries were (WAY; Sigma Chemical

(5 p.g/ml 5 .tg/ml

MA) + 50 tM

oocyte.

The

consisted cells. For

BSA (ICN insulin, selenium; 3-isobut

resulting

of the zona convenience,

oocytectomized pellucida we refer

as “intact follicles” throughout however, that the collagenase isolate the preantral follicles

(OOX)

follicle

surrounded by granulosa to the non-OOX follicles

this paper. It should be noted, treatment that was used to removes most of the theca cells

and components of the basal lamina. In addition, when these follicles are cultured, the granulosa cells attach to the collagen substratum and the oocyte develops within a stalk of granulosa cells that differentiate within a 10-day culture period however,

the

follicles

were

tissue MA) were as described

into functional [37]; in these cultured

for

culture plates coated with by Torrance

cumulus cells experiments,

only

5 days.

(Costar Corpora250 p.1 rat tail colet al. [39]. Intact

and OOX preantral follicles were cultured in 1 ml conditioned or oocvte-conditioned WAY/BSA/ITS/IBMX or without I p.g/ml ovine FSH-17 (NIDDK, Baltimore, in the collagen-coated wells for 4 days. Conditioned dium

was

grown uncoated

prepared

oocytes wells

by culturing

cumulus

in WAY/BSA/ITS/IBMX for 24 h. Freshly prepared

dium was supplied to the follicle ter the 4-day culture, 3H-thymidine Company, NEN Research Products, to each

culture

periments, Transwell-COL size)

and

Gollection

Follicles

undifferentiated

as described

crystallized

the

withdrawal of the lancing piin the holding pipette aspirated

well

intact were

for

an

additional

follicles (6-mm

co-cultured

with

the membrane so that contact

cell-denuded

fully

(1 oocyte/p.l) conditioned

in me-

cultures every 2 days. Af(5 p.Ci/ml; Du Pont Boston, MA) was added

or OOX membranes

on

of unwith MD) me-

24

h.

In some

ex-

were grown on Costar diameter, 3.0-p.m pore

denuded

oocvtes

that

with the follicles with the granulosa

were

or under cells was

prevented.

METHODS

of Preantral relatively

the oocytes were microsurgically removed from half the follicles as described previously [35, 37]. Briefly, each follicle was held with a micropipette using negative pressure. A lancing pipette was pushed through the complex and into

placed either the membrane

cells, preantral follicles were isolated 12-day-old (C57BL/6J X SJL/J)F1 mice mechanical

1) iso-

cultures.

MATERIALS

Inc.,

proliferation:

Aldrich Chemical Co., Milwaukee, WI) + 5 mg/mI collagenase (Worthington Biochemical Corp., Freehold, NJ). Isolated preantral follicles were washed four times in enzymefree medium and cultured individually for 24 h on 2% agarose as described previously [37]. After overnight culture,

Twenty-four-well lion, Cambridge, lagen prepared

that allows in response

and help to maintain the structural follicle [37]. This putative role of the

in proliferation

im-

hyaluronindicating

developmentally-reg-

factor expansion

by mouse

preor

spontaneous [32-34]. In ad-

cells to synthesize expansion in vitro,

a microsurgical

secreted

the

cell

prevent removal

1197

the holding pipette. Upon pette, the negative pressure

influence

observed population

a role

from

cumulus cumulus

oocyte can be removed from plex (oocytectomy), we found

cyte

play

cumulus expansion-enabling cells to undergo cumulus [35-37].

species

than in the more distant cells of evidence supports early ob-

may

of the

or

on the regulation of granuis the loss of association of

oocyte in situ appears to promote and progesterone production

separation

growth

either alone or gonadotro-

function. The oocyte might of granulosa cells since

pairs the ability of the ic acid and to undergo

ovary

fibroblast

potentially

the oocyte. It has been more frequently in the

ulosa cells nearest the [29-31]. An increasing

the

in many

could

studies in vitro

(1)

of

[18], somato(19, 20], trans-

factors, factors

proliferation

that

interpretation of these losa cell proliferation these cells with division occurs

that (EGF) (IGF-i)

-13 121], and

and (2) these other growth

granulosa

[24-281. Another

suggested

growth factor growth factor-i

CELL PROLIFERATION

GRANULOSA

changes in the cytoskeletal Nevertheless the results

have

synthesizes epidermal medin-C/insulin-like

PROMOTE

granulosa the ovaries enzymatic [38],

of and

with

the

dissociated in Co., St. Louis,

ImmunoBiologicals,

5 i.g/ml iron-saturated Collaborative Research I methylxanthine

(IBMX;

and

Culture

of Mural

Granulosa

Mural granulosa cells were obtained of the ovaries of 22-day-old mice that 48

h previously

with

5 LU eCG

Follicles were punctured both oocyte-cumulus cell

had

(Diosynth,

GelLc

from arnral follicles been injected 44Oss,

with 25-gauge needles complexes and clumps

Holland). releasing of mural

granulosa cells. The mural granulosa cells were collected in WAY/BSA/ITS and dispersed by being gently drawn in and out of a Pasteur pipette, and the cell suspension was centrifuged for then resuspended

3 mm at 250 X g. The granulosa in fresh medium and dispersed

cells were again as

VANDERHYDEN

1198 described concentrations

above.

The onto

cells were plated fetal bovine serum

at subconfluent (FBS)-coated Lab-

Tek chamber slides (4 chambers/slide; ville, IL) in 400 p.1 WAY/BSA/ITS/IBMX 24 h. After

this

period

Nunc, mc, Naperand incubated for

of equilibration,

the

serum-free

me-

ET Al..

air-dried.

Autoradiographs

were

prepared

using

Kodak

(Rochester, NY) NTB2 photographic emulsion and the emulsion-coated slides were exposed at 4#{176}C for 4 days. After developing, the labeled and unlabeled cells were counted using an Olympus bright-field microscope (40x objective).

dium was changed to fresh unconditioned or oocyte-conditi()ned medium with or without 1 p.g/ml of FSH. The cells were cultured for 24 h and then 3H-thymidine (5 p.Ci/ml)

In the granulosa cell monolayer experiments, at least 9 replicates were analyzed with a minimum of 1 000 cells counted per replicate. In experiments using complexes and prean-

was

tral

follicles,

the per

largest diameter. A minimum of 1 500 cells treatment. Each field counted contained

added

for

Collection

an additional

and

Culture

24 h.

of Ooc-pte-Gurnulus

Cell

plexes

Goinplexes Oocyte-cumulus

cell

complexes

were

isolated

day-old mice injected 44-48 h previously Complexes were isolated by puncturing of the ovaries with 25-gauge needles and were then washed three times

tioned

medium

under

oil

Statistical

beled)

counted

were performed labeling index

in the

fields

showing

were 1-4

was

determined

for

a minimum (the percentage each

field

cance of differences between each termined by Chi-square analysis

[35]. Intact (conwith or without or oocyte-condi-

differences

were

above.

was added to each group for a further 3 h.

as the

mean

as described

were

counted com-

Analysis

Experiments The thymidmne

the

or

cells

or follicles.

present

medium

3H-thymidmne were incubated

22-

in WAY/BSA/ITS/IBMX, in fresh medium. Com-

plexes were OOX as described previously trol) and OOX complexes were cultured FSH in 200-pA drops of unconditioned After 4 h, 50 p.Ci/ml and the complexes

from

with 5 IU eCG. the antral follicles

the

considered

variability ±

examined.

Signifi-

treatment group was desvi,th Yates’ correction;

significant

between

of three times. of cells la-

at p

experiments,

0.01.


to

‘0

“‘U)0.-

#{149},:.

0

U)OC

00

.2 CCD

C

a,g

A

‘.

‘-

.0

CD

2

U

-

U)

‘0

--

‘0

-e

E

-

5)

c C

-jCD

0

(0

9. = CD5)

C CD

-

:.

..

-

.. ‘

-

‘U

w 0

2 .9

U)

5,

-j

E.

0)

U)

a

CD

:..

::

CD

‘-‘0

-.,,:

#{149} 5:

0

OE

#{149}

I.

O#{149}

C

#{149} ‘C.,

..j#{149}-

,

CD

o

‘

, CD

CDU)

CD

I

p.

0

E,

C

‘

(0

E .c 0 ....

a o aCD

0

0

)

0 C

5)

o

CD

#{149}CCD

0

E

CD -

CD U)

0)

o

CD

CD U)

0

‘

cODE 0

Z

CD.C

_C

I

U)

0 U

_C

.

2-

00 CD .C’0

u.

.9 -

a

CD ‘0

1200

VANDERHYDEN

ET AL.

A

B

-.--_--...

‘I’

I-

..

-y

D FIG. 2. Autoradiographs IA) Intact follicle. IS) Intact in oocyte-conditioned

of preantral follicles grown on rat tail collagen follicle cultured in oocyte-conditioned medium.

medium.

All

of the

micrographs

are

the

same

In #{149}Intact

000x

U,

magnification;

Vitro

the

bar

Pro4feration

Antral To ulation mural

for 5 days and labeled with 3H-thymidine. (C) OOX follicle. ID) OOX follicle cultured

of Mural

C 0) C

m.

Granulosa

Cells from

investigate a possible role for the of proliferation of more differentiated granulosa cells were isolated from

oocyte in the reggranulosa cells, antral follicles and

for 48 h in unconditioned by denuded oocytes for

medium 24 h. Dur-

ing the labeling period (the latter 24 h of culture), 11.6 ± 0.6% of unstimulated cells had incorporated 3H-thymidine (Fig. 4). The presence of oocyte-conditioned medium resulted in a 41% increase (p < 0.01) in the thymidmne labeling index of mural granulosa cells. FSH also stimulated an increase in the thymidine labeling index of those cells

2

Control Oocyte-Condltloned

100

Follicles

cultured as a monolayer or medium conditioned

CD ‘5,

indicates

k._...

FIG. 3. Incorporation of ‘H-thymidine by complexes from preantral follicles during a 24-h incubation. Each bar represents the mean ± SEM of the labeling indexes (percentage labeled cells) for intact and OOX follicles in four treatment groups. There were at least 20 follicles in each group. Oocytectomy resulted in a decrease in the labeling index of preantral follicles in all culture conditions (p < 0.01). Oocyte-conditioned medium stimulated granulosa cell proliferation in both control and OOX complexes in either the absence or presence of FSH (p < 0.01).

to 14.9 ± hancement tioned

In Vitro Antral

1.4%

of

(p