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
is
Vol. 78, No. 3, 1977
BIOCHEMICAL
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