BIOLOGY OF REPRODUCTION 56, 1458-1465 (1997)
Insulin-Like Growth Factor-Binding Protein-2 and -3: Their Biological Effects in
Bovine Thecal Cells'
L.J. Spicer,2 R.E. Stewart, P. Alvarez, C.C. Francisco, and B.E. Keefer
Department of Animal Science, Oklahoma State University, Stillwater, Oklahoma 74078
ABSTRACT
INTRODUCTION
The insulin-like growth factor (IGF) system is composed
of IGF-I and -II, IGF receptors, and IGF-binding proteins
(IGFBPs) (for reviews see [1-3]). Both IGF-I and IGF-II
have been shown to be relevant promoters of ovarian follicular function of several species including cattle (for reviews see [3-5]). In most species, IGF-I stimulates mitogenesis of granulosa and thecal cells in the absence of gonadotropins, whereas in the presence of gonadotropins,
IGF-I stimulates steroidogenesis of granulosa and thecal
cells (for reviews see [3, 51).
It was more than 10 yr after the determination of the
amino acid sequence of IGF-I and IGF-II in 1978 [6, 7]
that the structure of an IGFBP and its mRNA in ovarian
Accepted January 22, 1997.
Received March 11, 1996.
'Approved for publication by the Director, Oklahoma Agricultural Experiment Station. This research was supported under project H-2088, project number HR4-032 from the Oklahoma Center for the Advancement
of Science and Technology, and by the Cooperative State Research Service, U.S. Department of Agriculture, under Agreement No. 93-372039023.
2Correspondence. FAX: (405) 744-7390;
e-mail: igfleo@okway.okstate.edu
MATERIALS AND METHODS
Reagents and Hormones
Reagents used were Dulbecco's Modified Eagle medium
(DMEM), Ham's F-12, insulin (bovine; 25.7 U/mg), pronase E, collagenase, hyaluronidase, DNase, and fetal calf
serum (FCS), all obtained from Sigma Chemical Company
(St. Louis, MO); bovine LH (USDA-bLH-B5, LH activity
2.1
NIH-LH-S1 U/mg; FSH activity < 1.0% by weight)
obtained from the National Hormone and Pituitary Program
(Baltimore, MD); recombinant human IGFBP-2 (31 000
molecular weight) obtained from Austral Biologicals (San
Ramon, CA); and recombinant human IGFBP-3 (47 000
molecular weight) obtained from Upstate Biotechnology,
Inc. (Lake Placid, NY).
Cell Culture
Ovaries were obtained at a nearby commercial abattoir
from beef and dairy cattle after slaughter. After transport to
the laboratory on ice (< 120 min), the ovaries were processed and thecal cells were obtained as previously described [17, 18]. Briefly, large ( 8 mm) follicles were dissected from the ovary, follicular fluid was aspirated, follicles were bisected, and each follicle wall was scraped and
1458
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This study was aimed at testing the hypothesis that the insulin-like growth factor-binding proteins (IGFBP)-2 and -3 can
modulate the hormone-dependent differentiation of thecal cells
in vitro. Thecal cells from large ( 8 mm) follicles were collected from cattle, cultured for 2 days in medium containing
10% fetal calf serum, washed, and then treated for an additional
2 days in serum-free medium with bovine LH (100 ng/ml), recombinant human insulin-like growth factor (IGF)-I (0 or 30
ng/ml), recombinant human IGFBP-2 (0, 200, or 400 ng/ml; i.e.,
0, 6.5, or 12.9 nM), or recombinant human IGFBP-3 (0, 200, or
400 ng/ml; i.e., 0, 4.3, or 8.5 nM). IGFBP-2 (200 and 400 ng/ml)
inhibited (p < 0.05) IGF-l-induced androstenedione production
by 18-30% but did not influence (p > 0.10) progesterone production or thecal cell proliferation inthe presence of LH and/or
IGF-I. In contrast, IGFBP-3 (200 ng/ml) inhibited the IGF-l-induced increase in thecal cell numbers by 76%, and thecal cell
progesterone and androstenedione production by 52% and
89%, respectively. A higher dose of IGF-I (i.e., 100 ng/ml) overcame the inhibitory effects of IGFBP-3 on IGF-l-induced cell
proliferation and on progesterone and androstenedione production by thecal cells. As with IGFBP-2, IGFBP-3 had no effect (p
> 0.10) on LH-induced progesterone or androstenedione production by thecal cells in the absence of IGF-I. Both IGFBP-2
and IGFBP-3 directly inhibited [12511GF-I and -II binding to thecal cells; IGFBP-2 was a weaker inhibitor of thecal [1251]IGF-I
and -II binding than IGFBP-3. These results indicate that
IGFBP-3 has a more pronounced inhibitory effect than IGFBP-2
on IGF-I action in cultured bovine thecal cells. Thus, IGFBP-3
may play a more significant role than IGFBP-2 in regulating thecal cell proliferation and steroidogenesis during follicular development in cattle.
tissue were first reported [8-10]. Since that time, the presence of several additional IGFBPs (i.e., IGFBP-1 through
-6) in the ovary of several species has been described (for
reviews see [3, 4, 11]). In particular, it appears that follicular fluid levels of IGFBP-2 decrease whereas IGFBP-3
remains unchanged as follicles enlarge and become steroidogenically active in swine, cattle, and sheep (for reviews
see [3, 5]). IGFBP-3 and IGFBP-2 are the first and second
most prevalent IGFBPs, respectively, in ovarian follicular
fluid of pigs, cattle, and sheep [3, 12-14]. In particular,
IGFBP-2 activity in follicular fluid is lower in the growing
dominant vs. subordinate follicles [13], whereas androstenedione and estradiol concentrations in follicular fluid are
greater in the growing dominant follicle vs. subordinate follicles early in the estrous cycle of cattle [13]. Whether the
lower steroid production by subordinate follicles is a direct
result of the greater IGFBP-2 levels is unknown. Although
previous studies have shown that IGFBPs can directly inhibit FSH-induced steroid production by rat granulosa cells
[10, 15, 16], previous investigations have not evaluated the
effect of IGFBPs on steroidogenesis of thecal cells in any
species or on mitogenesis of ovarian cells in any species.
Potential in vivo effects of IGFBP-2 and -3 could be classified as endocrine or paracrine as well as autocrine (for
review see [3]). Moreover, direct effects of IGFBPs on
ovarian follicular cell mitogenesis and steroidogenesis in
domestic animals have not been reported. Therefore, we set
out to test the hypothesis that IGFBP-2 and IGFBP-3 can
modulate proliferation and steroidogenesis of bovine thecal
cells in vitro.
THECAL CELLS AND INSULIN-LIKE GROWTH FACTOR-BINDING PROTEINS
IGFBP-3 on LH (dose dependent)-stimulated thecal cell
steroidogenesis. Theca cells were cultured for 2 days in
10% FCS and then cultured in serum-free medium for an
additional 2 days with bovine LH (0, 1, 3, 10, 30, or 100
ng/ml), IGFBP-3 (0 or 200 ng/ml), and insulin (100 ng/ml).
The doses of LH, IGFBP-3, and insulin were selected based
on previous studies [18] and as described for experiment 2.
Experiment 6 was conducted to determine whether
IGFBP-2 and IGFBP-3 inhibit the binding of [ 25 I]IGF-I or
[125 I]IGF-II to their receptors. Thecal cells were obtained
from large follicles, cultured for 3 days in 10% FCS,
washed twice with serum-free medium, and then used for
IGF-I binding assays. Binding of [' 2 5I]IGF-I and
[125 I]IGF-II to thecal cells was determined as previously
described [21, 26].
To further evaluate the relative potency of IGFBP-2 and
IGFBP-3 on competition for IGF-I binding, experiment 7
was conducted to determine whether IGFBP-2 or IGFBP-3
could inhibit binding of [ 25I]IGF-I to the IGF-I antiserum
in an IGF-I RIA. Various concentrations of IGFBP-2 and
IGFBP-3 were added to total binding tubes in the IGF-I
assay. The IGF-I assay was conducted as previously described [27].
Determination of Thecal Cell Numbers
Numbers of thecal cells were determined at the termination of experiments using a Coulter counter (Model Zm;
Coulter Electronics, Hialeah, FL) as previously described
[17]. Briefly, cells were exposed to 0.5 ml of trypsin
(0.25% [w:v] in 0.15 M NaCI) for 20 min at 25 0C and then
scraped from each well, diluted in 0.15 M NaCl, and enumerated.
Androstenedione RIA
Concentrations of androstenedione in culture medium
collected on Day 4 of culture were determined using solidphase RIA kits (ICN Biomedicals, Costa Mesa, CA) as previously described [18]. Intra- and interassay coefficients of
variation were 14% and 10%, respectively.
Progesterone RIA
Concentrations of progesterone in culture medium collected on Day 4 of culture were determined with an RIA
as previously described [17]. Intra- and interassay coefficients of variation were 16% and 20%, respectively.
IGF-II RIA
Concentrations of IGF-II in culture medium collected on
Day 4 of culture were determined in one RIA as previously
described [14]. Culture medium was concentrated 3- to
10-fold using Centricon-3 microconcentrators (Amicon,
Danvers, MA) prior to extraction. Assay sensitivity (i.e.,
90% of total binding) was 60 pg/tube, and intraassay coefficient of variation was 4.9%.
StatisticalAnalyses
Experimental data are presented as the least squares
means
SE of measurements for triplicate culture wells
from three or more experiments. Each experiment was performed with different pools of thecal cells collected from
approximately 30 ovaries for each pool. Main effects and
interactions on dependent variables (i.e., cell numbers and
steroid production) were assessed using General Linear
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flushed with Ham's F-12 to remove any remaining granulosa cells. The theca interna layer was microdissected from
the follicle wall and torn into small pieces. These pieces
were digested for 1 h at 37°C in Ham's F-12 containing 1
mg/ml of collagenase, 1 mg/ml of hyaluronidase, 1 mg/ml
of pronase E, and 0.01 mg/ml of DNase on a rocking platform shaker. After incubation, undigested tissue was removed from the cell suspension by filtration, and the dispersed cells were washed and resuspended in serum-free
medium [18]. The number of viable cells was determined
using the trypan blue exclusion method, and averaged 90
± 3% of total thecal cells. Contamination of thecal cells
by granulosa cells is minimal using this procedure (i.e., <
10% [19, 20]).
Medium was a 1:1 (v:v) mixture of DMEM and Ham's
F-12 containing 0.12 mM gentamicin and 38.5 mM sodium
bicarbonate. Approximately 2 x 105 viable cells in 45-110
Rl of medium were added to Falcon multiwell plates
(#3047; Becton Dickinson and Co., Lincoln Park, NJ) containing 1 ml of medium. Cultures were kept at 38.5°C in a
5% CO 2 atmosphere. To obtain optimal attachment, cells
were maintained in the presence of 10% FCS for the first
2 days of culture, with a medium change occurring on Day
1. After this time, cells were washed twice with 0.5 ml of
serum-free medium, and incubations were continued in serum-free medium with or without added hormones for an
additional 48 h (i.e., from Day 2 to 4 of culture) without a
change of media.
Experiments 1 and 2 were conducted to evaluate the
dose-response effect of IGFBP-2 and IGFBP-3, respectively, on the action of LH and IGF-I on thecal cell proliferation and steroidogenesis. Thecal cells were cultured for 2
days in 10% FCS and then cultured in serum-free medium
for an additional 2 days with 100 ng/ml of LH in the absence or presence of 30 ng/ml of IGF-I and either IGFBP-2
(0, 200, and 400 ng/ml; i.e., 0, 6.5, and 12.9 nM) or
IGFBP-3 (0, 200, and 400 ng/ml; i.e., 0, 4.3, and 8.5 nM).
The doses of IGF-I and LH were selected based on previous
studies [18, 21]. The 30 ng/ml dose of IGF-I was selected
as a dose of IGF-I that elicits a steroidogenic response so
that the IGFBP concentrations used would be 2-3 times
greater than IGF-I concentrations; this ratio has been reported to be effective in inhibiting IGF-I action [22, 23].
Concentrations of immunoreactive IGFBP-2 in blood of
cattle and women have been reported to be between 100
and 500 ng/ml [24, 25]. Concentrations of immunoreactive
IGFBP-3 have not been reported for cattle but average between 2 and 4 g/ml in humans [25].
Experiment 3 was conducted to evaluate the effect of
IGFBP-3 on IGF-I (dose dependent)-stimulated thecal cell
proliferation and steroidogenesis. Thecal cells were cultured for 2 days in 10% FCS and then cultured in serumfree medium for an additional 2 days with 100 ng/ml of
LH in the absence or presence of IGF-I (0, 30, or 100
ng/ml) and IGFBP-3 (0 or 200 ng/ml). The doses of IGF-I,
LH, and IGFBP-3 were selected based on previous studies
[18] and as described for experiments 1 and 2.
Experiment 4 was conducted to evaluate the effect of
IGFBP-2 on LH (dose dependent)-stimulated thecal cell
steroidogenesis. Thecal cells were cultured for 2 days in
10% FCS and then cultured in serum-free medium for an
additional 2 days with bovine LH (0, 3, or 10 ng/ml),
IGFBP-2 (0 or 200 ng/ml), and insulin (100 ng/ml). The
doses of LH, IGFBP-2, and insulin were selected based on
previous studies [18] and as described for experiment 1.
Experiment 5 was conducted to evaluate the effect of
1459
SPICER ET AL.
1460
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FIG. 1. Effects of IGFBP-2 on IGF-I-stimulated thecal cell proliferation
(A), progesterone production (B), and androstenedione production (C) by
thecal cells in the presence of LH (experiment 1). Thecal cells were cultured for 2 days in the presence of 10% FCS and then treated in serumfree media with 100 ng/ml of LH, IGF-I (0 or 30 ng/ml), and IGFBP-2 (0,
200, or 400 ng/ml) for an additional 2 days. Values are means from three
separate replicate experiments. Within a panel, means without a common
superscript differ (p < 0.05).
FIG. 2. Effects of IGFBP-3 on IGF-I-stimulated thecal cell proliferation
(A), progesterone production (B), and androstenedione production (C) by
thecal cells in the presence of LH (experiment 2). Thecal cells were cultured for 2 days in the presence of 10% FCS and then treated in serumfree media with 100 ng/ml of LH, IGF-I (0 or 30 ng/ml), and IGFBP-3 (0,
200, or 400 ng/ml) for an additional 2 days. Values are means from three
separate replicate experiments. Within a panel, means without a common
superscript differ (p < 0.05).
Models procedures of the Statistical Analysis System [28].
Each well was a replicate, and each experiment contained
three replicates per treatment. When steroid production was
expressed as nanograms or picograms per 105 cells per 48
h, cell numbers at the termination of the experiment were
used for this calculation. Specific differences in steroid production and cell numbers between treatments were determined using the Fisher's protected least significant difference procedure [29].
Experiment 2
RESULTS
Experiment 1
The dose-response effects of IGFBP-2 on IGF-I- and
LH-induced thecal cell steroidogenesis and cell numbers
are shown in Figure 1. In the absence of IGFBP-2, 30 ng/ml
of IGF-I increased (p < 0.001) thecal cell numbers by 1.4fold, and LH induced progesterone and androstenedione
production by 1.7- and 3.9-fold, respectively (Fig. 1). In
the absence of IGF-I, 200 and 400 ng/ml of IGFBP-2 had
no effect (p > 0.10) on cell numbers or on LH-induced
progesterone and androstenedione production. However,
200 and 400 ng/ml of IGFBP-2 decreased (p < 0.05) the
IGF-I-induced increase in androstenedione production by
30% and 18%, respectively. In contrast, 200 and 400 ng/ml
of IGFBP-2 had no effect on IGF-I-induced thecal cell proliferation or progesterone production (Fig. 1).
The dose-response effects of IGFBP-3 on IGF-I- and
LH-induced thecal cell steroidogenesis and cell numbers
are shown in Figure 2. In the absence of IGFBP-3, 30 ng/ml
of IGF-I increased (p < 0.01) thecal cell numbers by 1.3fold and LH-induced progesterone and androstenedione
production by 1.7- and 3.2-fold, respectively (Fig. 2). In
the absence of IGF-I, IGFBP-3 had no effect (p > 0.10) on
cell numbers or on LH-induced progesterone or androstenedione production. In contrast, 200 and 400 ng/ml of
IGFBP-3 inhibited (p < 0.05) the IGF-I-induced increase
in thecal cell proliferation, progesterone production, and androstenedione production by 71% and 41%, 63% and 66%,
and 81% and 51%, respectively (Fig. 2).
Experiment 3
To determine whether higher doses of IGF-I could overcome the inhibitory effects of IGFBP-3, we conducted studies summarized in Figure 3. In the absence of IGFBP-3, 30
and 100 ng/ml of IGF-I increased (p < 0.001) thecal cell
numbers and LH-induced progesterone and androstenedione production in a dose-dependent manner (Fig. 3). In the
absence of IGF-I, 200 ng/ml of IGFBP-3 had no effect (p
> 0.10) on cell numbers or on LH-induced progesterone
and androstenedione production. However, 200 ng/ml of
IGFBP-3 reduced (p < 0.05) cell numbers (by 56%) and
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200
400
IQFBP-2, ng/mL
THECAL CELLS AND INSULIN-LIKE GROWTH FACTOR-BINDING PROTEINS
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FIG. 3. Effects of IGFBP-3 (BP-3) on IGF-I-stimulated thecal cell proliferation (A), progesterone (B), and androstenedione (C) production by thecal cells in the presence of LH (experiment 3). Thecal cells were cultured
for 2 days in the presence of 10% FCS and then treated in serum-free
media with 100 ng/ml of LH, IGF-I (0 or 30 ng/ml), and IGFBP-3 (0 or
200) for an additional 2 days. Values are means from four separate replicate experiments. Within a panel, means without a common superscript
differ (p < 0.05).
androstenedione production (by 55%) stimulated by 30
ng/ml of IGF-I and 100 ng/ml of LH. In the presence of
100 ng/ml of IGF-I and LH, however, IGFBP-3 had no
effect (p > 0.10) on cell numbers or androstenedione production (Fig. 3). Basal and IGF-I-induced progesterone production was not significantly affected by IGFBP-3 (Fig.
3B), although IGF-I-induced progesterone was reduced by
31% by IGFBP-3; IGFBP-3 significantly inhibited IGF-Iinduced progesterone in two of the four experiments summarized in Figure 3B (data not shown).
Experiment 5
To determine whether IGFBP-3 could influence the stimulatory effects of low doses of LH on progesterone and
androstenedione production, we conducted studies summarized in Figure 5. LH caused a dose-dependent increase
10
FIG. 4. Effects of IGFBP-2 on IGF--stimulated progesterone (A) and androstenedione (B) production by thecal cells (experiment 4). Thecal cells
were cultured for 2 days in the presence of 10% FCS and then treated in
serum-free media with 100 ng/ml of insulin, IGFBP-2 (0 or 200 ng/ml),
and LH (0, 3, or 10 ng/ml) for an additional 2 days. Values are means
from three separate replicate experiments. Within a panel, means without
a common superscript differ (p < 0.05).
(p < 0.05) in both progesterone and androstenedione production by thecal cells (Figure 5). However, IGFBP-3 had
no effect (p > 0.10) on basal or LH-induced progesterone
or androstenedione production (Figure 5).
Experiment 6
To determine whether IGFBP-2 or IGFBP-3 inhibits
binding of [125I]IGF-I or [ 25 I]IGF-II to their receptors in
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Experiment 4
To determine whether IGFBP-2 could influence the stimulatory effects of low doses of LH on progesterone and
androstenedione production, we conducted studies summarized in Figure 4. Both 3 and 10 ng/ml of LH increased
(p < 0.01) progesterone and androstenedione production by
thecal cells (Fig. 4). However, IGFBP-2 had no effect (p >
0.10) on progesterone or androstenedione production induced by LH (Fig. 4).
3
LH, ng/mL
o
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LH, ng/mL
100
1000
800
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600
0
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400
200
0
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10
30 100
LH, ng/mL
FIG. 5. Effects of IGFBP-3 on LH-stimulated progesterone (A) and androstenedione (B) production by thecal cells (experiment 5). Thecal cells
were cultured for 2 days in the presence of 10% FCS and then treated in
serum-free media with 100 ng/ml of insulin, IGFBP-3 (0 or 200 ng/ml),
and LH (0, 1, 3, 10, 30, or 100 ng/ml) for an additional 2 days. Values
are means from three separate replicate experiments. Within a panel,
means with a common superscript differ (p < 0.05).
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1461
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SPICER ET AL.
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on subsequent specific binding of [ 25I]IGF-I thecal cells;
specific binding of [12 5I]IGF-I to thecal cells averaged 5330
109 cpm/105 cells for control and
± 103 and 5598
IGFBP-3-treated cultures, respectively (n = 5 separate experiments).
"' I-IGF-11
b
Experiment 7
b
Av
C
c:
25
5
FIG. 6. Effects of IGFBP-2 and -3 on [' 111GF-I and ['2111GF-II binding
to thecal cells (experiment 6). Thecal cells were cultured for 3 days in
the presence of 10% FCS, washed with serum-free media, and then treated with 0 or 200 ng/ml of either IGFBP-2, IGFBP-3, IGF-I, or IGF-II during
the binding assay. Values are means of three separate replicate experiments and expressed as a percentage of buffer control (0 ng/ml of IGFBP-2
or -3) incubations. a,b,,,d For each respective tracer, means without a comn\
in
li(ffr
-nn clnrc.rnt -UIIIJCI"I
...II,
I
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es summarized in Figure 6.
thecal cells, we conducted studied
As depicted in Figure 6, 20C ng/ml of IGFBP-2 and
IGFBP-3 inhibited (p < 0.05) [ 1251]IGF-I and [ 25 I]IGF-II
binding to thecal cells; IGFBP- 3 inhibited [ 25 1]IGF-I and
-II binding to a greater degree than did IGFBP-2. In cornparison, 200 ng/ml of IGF-I inlhibited (p < 0.05) specific
[1 25I]IGF-I binding to thecal ce lls, whereas 200 ng/ml of
IGF-I had a weak inhibitory efeffect on thecal [12 51]IGF-II
binding.
An additional set of experimients was conducted to determine whether IGFBP-3 treatnnent, rather than competing
for IGF-I binding, could alter t]he number of thecal IGF-I
receptors and thereby reduce the effectiveness of IGF-I.
Thecal cells were cultured for 2 days in 10% FCS and then
cultured for an additional 2 daays in serum-free medium
containing 10 ng/ml of insulin in the presence or absence
of 50 ng/ml of IGFBP-3. Cells were washed and the IGF-I
binding assay was conducted. Exposure of thecal cells to
50 ng/ml of IGFBP-3 for 2 day s had no effect (p > 0.05)
-
rh-IGF-I
Pg
°-- IcGFBP-2 .. ,' IGFBP-3
ng
ng
100
80
aI
60
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Us
40
20
0
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,
1
,
10
100
1000
pg or njg per tube
FIG. 7. Effects of IGFBP-2 and -3 on [['25111GF-I binding to the IGF-I antiserum in an IGF-I RIA. IGFBP-2 or IC FBP-3 was added to the assay at
0.5, 1.25, 5.0, and 10.0 ng/tube; recomibinant human IGF-I was added at
pg/tube. Values are means of
'
7.8, 15.6, 31.25, 62.5, 125, 250, and 500
duplicate determinations from a representative assay and expressed as a
percentage of total binding (0 pg/tube (of IGF-I).
DISCUSSION
Results of the present study revealed that 1) IGFBP-2
weakly inhibited IGF-I-induced androstenedione production by thecal cells but had no effect on IGF-I-induced thecal cell proliferation or progesterone production; 2)
IGFBP-3 (200 ng/ml) inhibited thecal cell numbers and androstenedione production induced by 30 ng/ml of IGF-I,
while addition of 100 ng/ml of IGF-I reversed this inhibitory effect; 3) IGFBP-2 and IGFBP-3 had no effect on
LH-induced progesterone and androstenedione production
by thecal cells in the absence of IGF-I; and 4) IGFBP-3
was a more potent inhibitor of [ 125I]IGF-I and-II binding to
thecal cells and to IGF-I antiserum than was IGFBP-2, but
IGFBP-3 had no effect on numbers of IGF-I receptors.
For the first time, the actions of IGFBP-2 and -3 on
thecal cell steroidogenesis have been evaluated. Previous
studies using cultured rat Leydig cells showed that recombinant human IGFBP-2 inhibited IGF-I-induced testosterone production but not hCG-induced testosterone production [23]. We observed that IGFBP-2 weakly inhibited IGFI-induced androstenedione production by bovine thecal
cells, whereas IGFBP-2 had no effect on LH-induced androstenedione production in the absence of IGF-I. In addition, we observed that IGFBP-2 had no effect on LHand IGF-I-induced progesterone production. Why IGFBP-2
had a significant effect on IGF-I-induced androstenedione
production but not progesterone production is unclear, but
a likely reason is that thecal cell androstenedione production is more sensitive to low doses of IGF-I than is progesterone production [18]. As with IGFBP-2, we found that
IGFBP-3 had no effect on LH-induced androstenedione or
progesterone production by bovine thecal cells. In contrast
to findings for IGFBP-2, we observed that IGFBP-3 reduced the stimulatory effect of IGF-I on progesterone and
androstenedione production and that these inhibitory effects
of IGFBP-3 were overcome with higher doses of IGF-I.
Previous studies have shown that purified porcine IGFBP-2
and IGFBP-3 and purified rat IGFBP-4 and IGFBP-5 can
inhibit, whereas recombinant human IGFBP-6 has no effect
on, FSH-induced estradiol and progesterone production by
cultured rat granulosa cells [10, 15, 16, 30]. In cultured human granulosa cells, purified human IGFBP-1 and recombinant human IGFBP-3 inhibited FSH-induced estradiol
and progesterone production and IGF-I-induced amino acid
uptake but had no effect on FSH-induced progesterone production [31-33]. In contrast, purified porcine IGFBP-3 in-
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BP-2 BP-3 IGF-I IGF-11
Treatment
0
To determine whether the potency of IGFBP-2 or
IGFBP-3 differed in terms of their ability to inhibit binding
of [ 25 1]IGF-I to the IGF-I antiserum in an IGF-I RIA, studies were conducted as summarized in Figure 7. IGFBP-3
at 5 and 10 ng/tube nearly completely blocked all binding
of [125 I]IGF-I to the IGF-I antiserum (Fig. 7). In contrast,
IGFBP-2 only weakly competed for [ 125 I]IGF-I binding; at
10 ng/tube, IGFBP-2 reduced binding to 60% of total binding. As estimated from linearized log-logit plots, IGFBP-3
and IGFBP-2 cross-reacted in the IGF-I assay at 2.9% and
0.2%, respectively.
THECAL CELLS AND INSULIN-LIKE GROWTH FACTOR-BINDING PROTEINS
ulosa and thecal cells in cattle both contain IGF-I mRNA
[3, 49], and IGF-II mRNA exists in thecal cells of sheep
[14] and rats [50], there has been no previous evidence for
bovine thecal or granulosa cell IGF-II production. We find
that bovine thecal cells produce - 1.5 ng of IGF-II/10 5
cells per 24 h and - 6 ng of IGF-I/10 5 cells per 24 h ([49];
unpublished results). Thus, IGFBP-2, with a greater affinity
to bind IGF-II than IGF-I [3, 11], may be inactivated by
IGF-II produced within the theca interna, allowing for the
effects of locally produced or systemic IGF-I to be manifested. Alternatively, IGFBP-2 may be selectively degraded
by bovine thecal cells, since porcine granulosa cells can
degrade IGFBP-3 [51] and rat granulosa cells can degrade
IGFBP-5 [52, 53]. Further research will be required to verify this suggestion. Moreover, the hormonal milieu within
the follicle may alter the biological effects of the IGFBPs,
since IGF-I stimulates the production of both IGFBP-2 and
IGFBP-3 [54] and attenuates IGFBP-3 degradation [51] in
porcine granulosa cells. A recent study has shown that
IGF-I but not LH increases IGFBP-2 and -4 production by
rat thecal cells in vitro [55]. In cultured bovine dermal fibroblasts, IGFBP-3 can prevent IGF-I-induced receptor
down-regulation, a process that renders cells refractory to
further stimulation by IGF-I [56]. Thus, numerous factors
may be involved in determining the cellular response to
IGFBPs and IGF-I.
Whether IGFBP-2 and IGFBP-3 are acting as endocrine,
paracrine, or autocrine regulators of ovarian follicular function in cattle is uncertain. IGFBP-2 and IGFBP-3 are the
predominant IGFBPs found in bovine [12, 13] and ovine
[14, 35] follicular fluid. Moreover, IGFBP-2 levels are
greater in small follicles than in peripheral blood, and they
decrease as follicles enlarge and become estrogen active in
sheep [14], cattle [12, 13], and humans [38, 39, 57]. Evidence suggests that IGFBP-2 is produced by porcine granulosa and thecal cells [36, 54, 58, 59] and human granulosa
cells [60] but not rat granulosa cells [15, 61]. Also,
IGFBP-3 appears to be produced by rat thecal cells but not
rat granulosa cells [15, 61-63] or granulosa or thecal cells
of preovulatory porcine follicles [59]. On the basis of ligand blotting of concentrated spent culture medium, we
find that there is no detectable IGFBP-3 or IGFBP-2 produced by culture bovine thecal cells (unpublished results).
Furthermore, using a charcoal exchange assay [27], IGF
binding protein activity is not detectable in spent thecal cell
culture medium (unpublished results). Collectively, these
results indicate that IGFBP-2 and -3 may be produced by
ovarian follicular cells of some species and thus may act
as autocrine or paracrine regulators of ovarian follicular
steroidogenesis during follicular growth and atresia. However, based on results of the present study, it appears that
IGFBP-2 and -3 may be acting as primarily endocrine regulators of ovarian follicular function in cattle. Regardless
of the source of IGFBPs, imbalances between IGFs and
IGFBPs, such as those that occur during severe feed restriction [64], may be involved in the pathology of reproductive inefficiency and therefore warrant further study.
ACKNOWLEDGMENTS
The authors thank the National Hormone and Pituitary Program (University of Maryland School of Medicine, Baltimore) for supplying bovine
LH and IGF-I antiserum (UB3-189), Wellington Quality Meats (Wellington, KS) for their generous donations of bovine ovaries, and Paula Cinnamon for her excellent secretarial assistance.
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creased hCG-induced progesterone accumulation by intact
rat follicles cultured for 24 h [34]; IGFBP-3 significantly
decreased hCG-induced estrogen production and attenuated
the inhibitory effect of hCG and IGF-I on follicular cell
apoptosis in this same study. Collectively, it seems that regardless of the species from which they are derived,
IGFBP-1 to -5 consistently inhibit FSH-induced estradiol
production by granulosa cells of rats and humans, whereas
the effects of IGFBPs on LH/hCG-induced granulosa-cell
steroidogenesis are inconsistent. Our studies further indicate that IGFBPs may have no effect on gonadotropin-induced steroidogenesis by thecal cells. In vivo, follicular fluid levels of IGFBP-2 and IGFBP-3 are negatively correlated
with estradiol and androstenedione in cattle [12, 13]. Also,
growing dominant follicles have lower IGFBP-2 than nongrowing static dominant follicles [13]. Moreover, levels of
IGFBP-2 and other lower molecular weight IGFBPs in follicular fluid increase as follicles become atretic and lose
their ability of produce estradiol, whereas IGFBP-3 levels
do not change in bovine [12], ovine [14, 35], porcine [36],
and human [37, 38] follicles. In contrast, others have reported that IGFBP-3 activity decreases during human follicle development [39]. Recently, IGFBP-3 has been shown
to inhibit the ability of FSH to suppress apoptosis in cultured rat follicles [40]. Thus, in vitro and in vivo data suggest that the presence of IGFBP-3 and increased IGFBP-2
in follicles undergoing atresia may hasten the atretic process by inhibiting androstenedione and estradiol production, and that IGF-I at physiological concentrations can attenuate this inhibitory effect.
In addition to effects on thecal cell steroidogenesis,
IGFBP-3 but not IGFBP-2 inhibited thecal cell proliferation. No previous reports have evaluated the effect of
IGFBPs on ovarian cell mitosis or proliferation. Because
our steroid production data were corrected for the decrease
in cell numbers, it is unlikely the decrease in steroid production was due indirectly to a decrease in cell numbers.
Previously, IGFBP-2 and/or IGFBP-3 have been shown to
inhibit IGF-I-induced mitosis of cultured fibroblasts [41,
42], rat osteoblasts [43], and estradiol-stimulated breast
cancer cells [44]. However, other studies have reported that
IGFBP-2 and/or IGFBP-3 can enhance IGF-I-stimulated
mitosis of some reproductive and endocrine cells such as
fetal rat islets of Langerhans [45], human breast cancer cells
[46], and prostate carcinoma cells transfected with androgen receptor cDNA [47]; nontransfected prostate cells were
not affected by IGFBP-3 [47]. Collectively, these studies
along with those reviewed by Jones and Clemmons [2] suggest that numerous factors may interplay to determine the
response of a particular cell type to IGFBP-2 or IGFBP-3.
The mechanism by which IGFBP-2 and IGFBP-3 antagonize the stimulatory action of IGF-I on follicular mitogenesis and steroidogenesis in vivo is unclear; however, on
the basis of the present and previous [48] in vitro studies,
it is reasonable to propose that the mechanism involves the
binding of systemic and endogenously produced IGF-I. Results from our studies clearly indicate that IGFBP-3 blocks
IGF-I binding to its receptors and competes for [125I]IGF-I
binding to IGF-I antiserum in an IGF-I RIA, but 2-day
treatment with IGFBP-3 in vitro has no effect on the number of IGF-I receptors in thecal cells. Also, IGFBP-2
blocked IGF-I binding to its receptors to a lesser degree
than IGFBP-3 and weakly competed for [12 5I]IGF-I binding
to IGF-I antiserum in the IGF-I RIA, suggesting that the
affinity of thecal IGF-I receptors for IGF-I is greater than
the affinity of IGFBP-2 for binding IGF-I. Although gran-
1463
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SPICER ET AL.
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