BIOCHEMICAL

Vol. 78, No. 3, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

APPEARANCE OF LH-RECEPTORS AND LH-STIMULABLECYCLICAMP ACCUMULATION IN GRANULOSA CELLSDURINGFOLLICULARMATURATIONIN THE RAT OVARY A. Nimrod, E. Bedrak* and S.A. Lamprecht Department of HormoneResearch, The Weizmann Institute of Science, Rehovot, Israel Received

August

22,1977 SUMMARY

The number of receptors for humanchorionic gonadotropin (hCG) was low in granulosa cells (GC) of follicles at 9.00 h on the day of metestrus (530 f 150 sites/cell; mean % S.E.) and gradually increased thereafter to reach a maximum at 21.00 h on the day of proestrus (24,300 + 700). There was no significant difference in 1251-hCGbinding between GC collected before the preovulatory LH-surge and cells collected 7 h after the surge. LH-stimulable cyclic AMP accumulation was higher in GCcollected 7 h after the surge than in GCcollected before the surge or in GC from animals in which the LH-surge was blocked by treatment with Nembutal. Exogenous hCG (50 III/rat) also failed to induce desensitization of LH-stimulable adenylate cyclase in GCcollected 10 h after the injection. The results show (i) cyclic changes in the LH-receptor population of rat GC compatible with the concept that the receptor is induced by the estrogen and FSH surges of the preceding cycle, and (ii) failure of the preovulatory LHsurge to induce refractoriness of adenylate cyclase in rat GCin vivo. The maturation of the Graafian follicle of receptors

(1) for luteinizing

the action of the ovulatory to be under the control

is associated with the appearance

hormone (LH), which prepares the follicle

LH-surge.

of follicle

Induction of the LH-receptor is believed

stimulating

hormone (FSH), since it has

been shown that FSH induces LH binding sites in granulosa cells preantral

follicles

both in vivo

(2) and in vitro

gonadotropin (hCG) binds specifically

for

(3).

(GC) of rat

Humanchorionic

to the LH-receptor of rat ovaries (4).

Although previous studies of binding of labeled hCG to isolated porcine GC have shown that LH-receptor concentration

precise correlation not hitherto

increases with follicle

between LH-receptor levels and follicular

size (l),

a

development has

been demonstrated.

Exposure of preovulatory

follicles

been shown to render them refractory * Present address: Beersheva, Israel.

in

vitro

to further

Department of Biology,

to a high dose of LH has stimulation

by LH (5-B).

Ben-Gurion University

of the Negev,

Vol. 78, No. 3, 1977

It

BIOCHEMICAL

has been reported

of LH (9-12). yet

sites

involved

unclear,

Graafian

ation.

surge,

follicle

cyclic

occurs

this

diverge

the

appearance

was quantitatively both

the

binding

AMP accumulation

in an attempt

refractoriness

also

underlying

and opinions

study,

In addition,

stimulable

process

in

vivo

desensitization whether

after

the

of the

or not

a loss

surge follicle

of receptor

(8,10,13).

In the present tact

this

The mechanism

to LH is is

that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to resolve

to the hormone

of the

LH-receptors

analysed

during

follicular

and the

activity

of LH-

in relation

to the

of 1251-hCG of GC were

the

question

measured whether

on GC of in-

the LH-surge

matur-

LH-

induces

in GC in vivo. MATERIALS

AND METHODS

Animals. Threeto four-month-old Wistar-derived female rats (170-210 g) of the departmental colony were housed in air-conditioned quarters illuminated between 05.00 and 19.00 h. Only animals that had at least two consecutive normal 4-day cycles, as determined by daily vaginal smears, were used. Pentobarbitone sodium (Nembutal, Abbot, 6 mg/rat) or hCG (Pregnyl, Organon, 50 Ill/ rat) were administered intraperitoneally at 14.00 h on the day of proestrus where indicated. Cell collection. Animals were killed at specified times by cervical dislocation, and the ovaries were excised and immersed either in ice-cold buffered sucrose solution for binding studies (14) or in Krebs-Ringer bicarbonateglucose buffer for adenylate cyclase assay (15). Granulosa cells were isolated by puncture of the largest follicles (4-7 follicles per ovary) and gentle expression into medium using a dissecting microscope (15). Cells derived from 3 animals were combined, sedimented by centrifugation at 250 x g for 8 min at 4fC and resuspended in an appropriate volume of buffer for 1251-hCG binding studies or for the assay of LH-stimulable cyclic AMP accumulation. Iodination of hCG (13,500 units/mg; Serono, Italy Assay of hCG binding. was carried out by the lactoperoxidase method described by Miyachi et al. (16) The biological activity of the labeled preparation was determined by assay in an adenylate cyclase system (17) and used to determine the mass of active radioactivity of labeled hCG was hormone. Based on this mass, the specific found to be 20-50 uCi/Pg. Binding of 1251-hCG to granulosa cells was assayed Briefly, cell suspensions (50 Pl containas described by Nimrod et al,. (3)s ing 5-8 x lo4 cells) were incubated for 3 h at 37’C in a shaking incubator together with 50 ~1 of buffer containing 11 rig/ml of 1251-hCG and varying Nonspecific binding was determined by incubating amounts of unlabeled hCG. cells in the presence of a ZOO-fold excess of unlabeled hCG, and this value was substracted from all values of bound radioactivity prior to the calculation of binding parameters (14). LH-stimulable CAMP accumulation. Cell suspensions (2-5 x lo5 cells/ml) were incubated with shaking at 37’C for 20 min in Krebs-Ringer bicarbonate buffer (0.4 ml) containing-l mg/ml glucose and 0.1 mg/ml of 3-isobutyl-lmethyl-xanthine (IBMX) . Some samples also contained various amounts of ovine

978

BIOCHEMICAL

Vol. 78, No. 3, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

hCG

Bound

(rig/ml)

Figure 1. Scatchard-plot analysis of lz51-hCG binding to granulosa cells obtained from rat follicles at different stages of the estrous cycle. Cell suspensions (0.1 ml; 5 x lo5 cells/ml) were incubated in triplicate with 1251-hCGand varying amounts of unlabeled hCG. Each line is derived from incubation of aliquots of a cell suspension obtained from 3 rats. The number of binding sites per cell, obtained from the intercept of the lines with the abscissa, was 2,500, 5,570 and 11,400 sites/cell for metestrous, diestrous and proestrous rats, respectively.

LH (NIH-LH-S18, kindly made available by the NIAMDD, N.I.H., Bethesda, Md.). The reaction was stopped by addition of.0.4 ml of 0.05 M sodium acetate buffer, pH 4.0 (S), and boiling for 5 min. Samples were then frozen and kept at -2O'C. Cyclic AMP content was assayed by the method of Gilman (18). RESULTS Binding of 1251-hCGto GCat various stages of the estrous cycle.. Graafian follicles

exhibited

specific

GC of

binding of lz51-hCG, with an apparent

association constant (Ka) of about 10lo M-l, based on a molecular weight of 47,000 daltons (Fig.

1).

The number of binding sites per cell,

from Scatchard plots (Fig.

I),

as determined

was low in the morning of the day of metestrus

(530 + 150 sites/cell;

mean It. S.E.;

metestrus and diestrus,

then increased steeply en proestrus,

maximum(24,300 + 700) at 21.00 h.

Fig. 2), gradually

increased during

A small, yet significant

to reach a (p < 0.05)

decline in the number of binding sites (15,000 +_3,200) was observed in GC collected

at 24.00 h on the day of proestrus (Fig. 2).

979

Vol. 78, No. 3, 1977

BIOCHEMICAL

I200

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I200L!O( 2L

2400

METESTRUS

DIESTAUS Days

of

PROESTRUS

Cycle

Figure 2. Appearance of hCG binding sites in granulosa cells of the developing ovarian follicles during the estrous cycle of the rat. The bars and vertical lines indicate the mean + S.E. of the number of binding sites per cell obtained by Scatchard-plot analysis, as described in the Method section. The figures in brackets indicate the number of cell suspensions used, each obtained from 2-3 rats.

Rate of cyclic LH-surge.

In two sets

follicles

accumulation

increased set

assayed

injected

dose)

AMP accumulation stinulable

after

activities

Nembutal-treated LH-stinulable with

in Table

adenylate

animals

served

to block

the finding surge, cyclase of hCG

in follicular

the hormone. 980

were

the viz.

1, confirmed

cyclase

the peak

as a control,

refractoriness

Administration

from preovulatory of the after

cyclic

AMP

divided

into

1).

to induce

of GC adenylate rats.

(Table

hCG (50 Ill/rat;

the endogenous

collected

and LH-stimulable

in order

h with

in an attempt

given

basal

one group

at 14.00

the endogenous

one h before

proestrous

Nembutal

and after

3 h and 7 h, respectively,

LH-surge

of experiments,

h with

was treated

The results,

the

were

about

h, i.e. Both

concurrently:

at 14.00

ovulatory

(19). after

cells

h (i.e.

and 24.00

LH-surge

In a third groups

at 16.00

and at 21.00

the peak of the

in GC before

of experiments,

on proestrus

LH-surge),

group

AMP accumulation

since were

another

LH-surge, ZO-times

3

group

was

and a third the minimal

in the GC in vivo. of increased both higher

in vivo GC within

the

basal in control

failed

cyclic and LHthan

to desensitize

10 h after

treatment

in

Vol. 78, No, 3, 1977

BIOCHEMICAL

I'able

1. LH-stimulable CAMP accumulation ovulatory follicles of proestrous

:xperiment No.

Time of Sacrifice (h)

*

**

Treatment

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

by granulosa cells isolated from prerats. IBMX (100 rig/ml) was added.

..*- ac cumulation* cfww (pmol/tube/20 ovine LH -.- (tkVm1)

CAMP content* i~~~~r-~ion

0

0.1

min)

0.4

10.0

(pmol/tube)

1

Untreated

15.30 21.00 24.00

2.5 3.5 4.5

? 0.3 k 0.3 i 0.3

2.5 4.0 6.0

t 0.3 + 0.2 k 0.5

2

Untreated

15.30 21.00 24.00

4.5 2 0.2 6.8 f 0.5 9.0 f 1.4

4.2 9.0 13.4

c 0.2 + 0.1 f 1.7

3

Untreated Nembutal** hCG**

24.00 24.00 24.00

2.0 + 0.1 1.3 f 0.1 2.2 f 0.2

2.9 1.2 4.7

? 0.4 2 0.1 + 0.8

12.7 30.9 41.8

9.6 3.8 11.6

+ 1.7 + 1.6 + 5.8

2 2.5 2 1.0 + 3.4

13.5 13.5 24.0

i 2.5 i 2.0 + 2.0

14.8 34.7 46.5

2 2.7

11.3 t 3.5 5.1 c 1.0 12.4 zt 4.8

by 3 cell suspensions, Values represent the mean ? S.E. of CAMP accumulation Granulosa cell suspensions were incubated in duplicate, derived from 3 rats. CAMP content in each sample (cells plus medium) was assayed in duplicate as described in the Methods section. Nembutal

(6 mg/rat)

and hCG (50 IU/rat)

were

given

intraperitoneally

each and

at 14.00

DISCUSSION Several increase sults

authors

in the are

precise

number

time-sequence in the

rat.

and FSH (2,3,20). and exposure

maximal

number

in the

adult

rat:

these

appearance

follicular

sites

in GC (1). and,

of LH-receptors

to FSH before

may take

a steep

rise

place

attained during

in the number

981

are

in addition, sites

an

action after

induced

re-

provide

during

required

(17,20)

before

about

cyclic

maturation

of 1251-hCG

is

The present

sequential

of 48 h is

LH-receptors

there

in GC of immature

by the

are not

control

growth

of GC binding

time-lapse

sites

during

observations

shown to be controlled

of binding

hormonal

receptor

Induction

A minimal

priming

A similar

with of the

has been

that

of LH-hCG

in accordance

maturation follicles

have reported

k 1.8 zt 5.3

a

follicular rat of estrogen estrogen and

72 h (20).

binding

of follicles sites

is

h.

BIOCHEMICAL

Vol. 78, No. 3, 1977

observed

(Fig.

estrous

2) in GC of follicles

cycle

to elevated

followed

by a FSH-surge

increase

in ovarian

between

10.00

to the

hormone-free

adenylate

endogenous from medium

absence

of a detectable

sistent

with

responsive

at that

time:

LH-surge,

ings ation

in agreement

changes

in LH sensitivity

that factor,

which

that

3 h after

ex-

LH, however,

AMP accumulation

in

response sites

in

to LH in the viva

is

in several

of receptor

of available that

surge

con-

hormone-

just

prior

from

It

(9,lO) may thus

of desensitization

is

suggested

to the

(Fig.

surge

increased

suggesting

a lack

of desensitiz-

PMSG-treated

rats

with

reports

(Prof.

(11,lZ)

be that

refractoriness

adenylate

other

on the formation cell

These

find-

K. Ahren,

of preovulatory

and rat

components

2).

in GC fol-

1).

cyclase of the whole

than

the GC, or

of an inhibitory

collection. by the report

of adenylate

982

in GC 7 h

(Table

is dependent

explanation

significant-

was not

markedly

of follicular

away during

sites

respect

4 h earlier,

of the hormone data

to LH

hCG with

LH receptors

observed

in contrast

of rabbit

has been washed

the rate

the number

measured

are

become refractory

and to exogenous

AMP accumulation

desensitization

An alternative (13)

but

the behavior

follicular

that

preliminary

follicles.

reflects

surge

than

LH in GC isolated

communication),

follicle

a steep

was observed

of bound

described

GC do not

is

endogenous with

personal

Graafian

that

lower

cyclic

to the

to endogenous

of intact

receptors

the number

from

LH-stimulable

are

of cyclic

receptor

nor

although

different

exposure

An effect

in available

to the preovulatory

ly decreased

lowing

follicles

was unchanged

biological

indicate

activity,

Moreover,

rate

of "spare"

cyclase

significantly

2).

Likewise,

(11).

The observed

decrease

results

exposure

the

in the

1).

to adenylate

after

(Fig.

21)

(23).

The present following

LH-surge

22).

in rat

sites

in the preceding

to mid-proestrus;

h of proestrus

hCG binding

the existence tissues

(mid-diestrus

activity

and 10.00

(Table

exposed

to mid-estrus;

cyclase

the rise

have been

levels

(mid-proestrus

of available

can be inferred

which

estrogen

h of diestrus

The number posure

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cyclase

of Conti

et al.

activity

by exo-

BIOCHEMICAL

Vol. 78, No. 3, 1977

genous jected. after

our

vitro

vitro

a later ever, lowing

rat

failure

complicate ovulation,

of follicles

in observing

to lower

experiments event.

was dependent

50% desensitization

and 10 h after

in vivo

ovaries

desensitization

h, but

LH-surge

exposed in

in 12-24

be that the

hCG in luteinized

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

of the time

interpretation thus

concentrations

beyond

of the

undergoing

results,

extensive

from

6 h (8).

than onset that

those

the

fact

It

that

applied

since

here

may well

exposure

to

GC are

in the

of refractoriness selected

in-

of LH is maximal

in GC 7 h after

to hCG derives

therefore

doses

within

desensitization

hormone

Extension

to high

is attained

exposure

and that

on the dose of hormone

in would,

the GC luteinize

vivo howfol-

differentiation.

Acknowledgements We are grateful to Mrs. Genia to Dr. Yoram Salomon for assay of hCG preparation, and to Dr. Uriel discussions. The work was supported Ford Foundation and the Population

Acevedo for skilful technical assistance, the biological activity of the iodinated Zor and Dr. Hans R. Lindner for helpful by a grant (to H.R. Lindner) of the Council, N.Y.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Channing, C.P. and Tsafriri, A., Metabolism, 5, 413 (1977). Zeleznik, A.J,, Midgley, A.R,, Jr. and Reichert, L-E., Jr,, Endocrinology, 95, 818 (1974). Nimrod, A., Tsafriri, A. and Lindner, H.R., Nature, 267, 632 (1977). Koch, Y., Zor, U., Chobsieng, P., Lamprecht, S.A., Pomerantz, S. and Lindner, H-R., J, Endocr.,+, 179 (1974). Lamprecht, S.A., Zor, U., Tsafriri, A. and Lindner, H.R., J. Endocr. 57, 217 (1973). S.A., Misulovin, Z., Koch, Y. and Lindner, H-R., Zor, U., Lamprecht, Biochim, Biophys. Acta, 428, 761 (1976). Hunzicker-Dunn, M. and Birnbaumer, L., Endocrinology, 99, 185 (1976). Lamprecht, S.A., Zor, Il., Salomon, Y., Koch, Y., Ah&n, K. and Lindner, H.R., J. Cyclic Nucleotide Res., 3, 69 (1977). Marsh, J.M., Mills, T.M. and LemaiFk, W.J., Biochim. Biophys. Acta, 304, 197 (1973). Mills, T.M. and McPherson, J.C. III, Proc. Sot. Exp. Biol. Med., 145, 446 (1974). Hunzicker-Dunn, M. and Birnbaumer, L., Endocrinology, 2, 198 (1976). Nilsson, L., Hillensj0, T. and Ekholm, C. Acta Endocr., (in press). Conti, M., Harwood, J.P., Hsueh, A.J.W., Dufau, M.L. and Catt, K.J., J. Biol. Chem., 251, 7729 (1976). Nimrod, A., Erickson, G.F. and Ryan, K.J., Endocrinology, 98, 56 (1976). Nimrod, A., Mol. Cell. Endocr., (in press). Miyachi, Y., Vaitukaitis, J.L., Nieschlag, E. and Lipsett, M.P., J. Clin. Endocr. Metab., 34, 23 (1972).

983

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Salomon, Y., Yanovsky, A., Mintz, Y., Amir, Y. and Lindner, H.R., J. Cyclic Nucleotide Res 18. Gilman, A.G., Proc . Nat,' . 'A% . p~~~s)* .', 67, 305 (1970). A., Lindner, H.R., Cordova, T. and Harell, A., 19. Ayalon, D., Tsafriri, J. Reprod, Fert., 31, 51 (1972). 20. Richards, J.S., Ireland, J-J., Rao, M.C., Bernath, G.A., Midgley, A.R., Jr. and Reichert, L,E,, Jr,, Endocrinology, 99, 1562 (1976). 21, Brown-Grant, K., Exley, D. and Naftolin, F., J. Endocr., 2, 295 (1970). 22, Daane, T.A. and Parlow, A.F,, Endocrinology, &, 653 (1971). 23. Catt, K.J. and Dufau, M.L., Ann. Rev. Physiol., 39, 529 (1977). 17.

984