Vol. 1, No. 1
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Different action of ovine GH on porcine theca and
granulosa cells proliferation and insulin-like growth
factors I- and 11-stimulated estradiol production
Jolanta Kolodziejczyk: Ewa L. Gregoraszczuk' ', Haim Leibovich3,Arieh Gertle13,
Zaboratory of Domestic Animal Physiology and Toxicolosy Department of Animal Physiology,
Institute of Zoology, Jagiellonian University, Krakokow, Poland; 'Institute of Biochemistry, Food Science
and Nutrition, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel
Received 15 July 2000; accepted 25 October 2000
SUMMARY
Growth hormone (GH) and insulin-like growth factors (IGFs) are recognized as regulators of
ovarian function. This study was designed to compare the effect of G H and IGFs added alone or
together on porcine theca interna and granulosa cells proliferation and steroidogenesis. Moreover,
the effect of G H on IGF-I secretion was examined. Cells were isolated from medium size follicles
and cultured bz vvitro for 48h in serum free medium. Estradiol and IGF-I medium concentrations
were determined by radioimmunoassays. Proliferation was evaluated by alamar blue assay and by
radiolabelled thymidine incorporation. G H increased IGF secretion by granulosa cells while
decreased its secretion by theca cells. Proliferation of both cell types was stimulated by IGF-I and
IGF-I1 (30 ngiml) and modestly inhibited by GH (100 ngiml). Insulin-like growth factor I1
increased, in a statistically significant manner, estradiol secretion by both cell types, while IGF-I
stimulated estradiol secretion to a greater extent by granulosa then by theca cells. The synergistic
action of G H and IGFs on estradiol secretion was stimulatory in theca cells and inhibitory in granulosa cells. These data demonstrate that despite its direct action on estradiol secretion by granulosa and theca cells, GH also modulated estradiol secretion induced by IGFs. Differences in the
estradiol production in response to GH alone and the effect of the synergistic action of G H and
IGFs suggest that different cellular mechanisms for these hormones are triggered in each cell type.
Key words: pig, theca cells, granulosa cells, GH, IGF-I, IGF-11, proliferation, estradiol.
INTRODUCTION
In mammalian species both female and male endocrine functions of gonads are regulated primarily by two pituitary gonadotropins: luteinizing hormone (LH) and follicle-stimulating hormone
(FSH). However, there is considerable evidence 14,281 that, in addition to these regulatory inputs,
ovarian function can be influenced by growth hormone (GH) either directly or via stimulation of
systemic andlor local production of the insulin-like growth factors (IGFs). It has been suggested
that the effect of G H in the ovary is mediated by a mechanism involving an elevation of IGF-I
level 1171. In humans [18] treatment with GH was associated with a rise in circulating concentrations of IGF-I. In pigs treatment with GH caused an increase in concentration of IGF-I in follicular fluid [30]. In addition, Samaras et al. [26] suggested that porcine ovarian cells produce both
IGF-I and IGF-11.
Previously, we have shown that G H action on estradiol secretion by porcine follicular cells was
tissue dependent [Ill. The question arises if the observed effect could be due to: 11 the influence
of G H on IGFs production by follicular cells, 21 the effect of G H on cell proliferation. In addition,
the effect of G H on basal and IGF I- and 11- stimulated estradiol secretion by theca and granulosa
cells was tested.
' Corresponding author: Labo~.atoryof Dolncstic Animal Physiology and Toxicolo&y,Department of Animal Physiology, Institute
of Zoology, Jagiellonian University, ul. Ingardena 6, 30-060 Krakrw, Poland; e-mail: [email protected]
Copyright 0 2001 by the Polish Society for Biology of Reproduction
34
GH and IGFs regulation o f porcine ovarian cells
MATERIALS AND METHODS
Reagents
Parker medium (M199), calf serum, trypsin and PBS were obtained from Biomed (Lublin, Poland).
Antibiotic-antymycotic solution x100, testosterone and bovine serum albumin (RIA-grade) were
purchased from Sigma Chemical Co., (St. Louis, MO, USA). Ovine growth hormone (oGH) was
prepared in the Institute of Biochemistry, Food Science and Nutrition (Rehovot, Israel) and
insulin-like growth factor I and IS were obtained from Dr. John Byatt from the Monsanto Co.
(St. Louis, MO, USA). Rabbit antiserum for IGF-I (UB2-495) was obtained from the Hormone
Distribution Program of NIDDK through the NIH National Hormone and Pituitary Program.
Cell cultures
Porcine ovaries obtained from a local abattoir were collected into a bottle filled with sterilized iced
saline and transported to the laboratory. Approximately 15 min elapsed from slaughter to ovary
collection. In each experiment six ovaries from three animals were selected for cell preparation.
Since each ovary yielded 4-6 follicles, the total number of follicles varied between 24 to 36 per
experiment. Medium follicles were obtained from ovaries collected on days 10-12 of the oestrus
cycle as described previously [lo, 221. This procedure was chosen to minimize possible variation
existing between follicles and animals. Granulosa (Gc) and theca interna (Tc) layers were subsequently prepared. The separation of Gc from the theca layer was performed according to the technique described by Stoklosowa et al. [31].
Briefly, Gc were scraped from the follicular wall with round-tip ophthalmologic tweezers and
rinsed several times with PBS. After collection, Gc were washed three times in M199 and were
recovered by centrifugation (10 min at 200xg). Viable granulosa cells (92%), determined by trypan
blue exclusion test, were finally suspended in 24-36 ml of M-199 medium supplemented with 5%
calf serum, yielding 1 ml of suspended cells per follicle. Then the cells were plated in I-ml well of
24 well plastic cell-culture plates (Falcon, Lincoln Park, NJ).
The theca cells from the same follicles were prepared as previously described in detail by
Stoklosowa et al. [31]. Briefly, the theca layers were placed in a drop of saline under the dissection
microscope. Theca interna was manually separated from the underlying theca externa. Isolated
theca interna tissue was then washed, cleaned, cut with scissors and exposed to trypsinization (10
min, 37°C) with 6-7 ml0.25% trypsin in PBS. The cells were separated by decantation and the procedure was repeated three times. Finally the cells were spun and resuspended in 24-36 ml of M-199
medium supplemented with 5% calf serum, yielding 1 ml of suspended cells per follicle and plated
as described above. The cell viability, using the trypan blue exclusion test, was 85%. The cultures
were maintained at 37°C in humidified atmosphere of 5% COz.
Experiment 1
This experiment was conducted to examine the influence of exogenous oGH (100 ngiml) on IGF -I
secretion by theca and granulosa cells. Cells were initially cultured in serum-containing M199 (5%
calf serum) for 24h to allow cell attachment to the plates. After 24h, serum-containing M199 was
discarded and cells were cultured for an additional 48h in MI99 supplemented with 0.1% BSA
1331. The concentration of oGH was established in our previous experiment Ill]. IGF-I level in
conditioned medium was determined [6] by radioimmunoassay (RIA). Intra- and inter-assay coefficients of variation were 8.7% and 12.4%, respectively. Every treatment was repeated four times.
Prior to the estimation the conditioned medium (0.1 ml) was concentrated by lyophylization, dissolved in 0.1 ml Et-OH acidic buffer, incubated for 30 min at room temperature, spun down, neutralized with 0.1 ml Tris buffer and 0.05 ml was taken for RIA. IGF-I concentrations are expressed
relative to a reference standard of recombinant human IGF-I.
Experiment 2
The study was conducted to demonstrate the role of ovine G H and IGFs in the stimulation of cells
proliferation. Cells were initially cultured in serum-containing M199 (5% calf serum) for 24h to
allow cell attachment to the plates. After 24h, medium was discarded and cells were cultured for
an additional 48h period in MI99 supplemented with 0.1% BSA. The doses of oIGF-I (30 ngiml)
and oIGF-I1 (30 ngiml) were used according to Veldhuis et al. 1341 and Spicer & Steewart [30].
Kolodziejczyk et al.
35
Ovine GH, oIGF-I and oIGF-11 alone or in combination of GH+IGF-I or GH+IGF-11 were
added at beginning of culture. Granulosa and theca cell proliferation was measured after 48h of
culture. Every treatment was conducted in four wells and each experiment was repeated three
times. The effect of oGH and oIGFs on granulosa and theca cells proliferation was conducted
using the following two independent assays:
s'/Al'amai-Blueassay. This assay is based on detection of metabolic activity. Alamar Blue contains
an oxidation-reduction indicator. Cellular proliferation induces chemical reduction of the media
with addition of Alamar Blue, which results in a change in colour from blue to red. The intensity
of red colour (reduced form of indicator) reflects the extent of cellular proliferation. The
absorbance was measured at a wave of 570 and 600 nm in micro ELISA plate reader (BIO-TEK
Instruments). The stock solution of Alamar Blue was aseptically added after 24 hour post-culture
set-up to culture wells in an amount equal to 10% of the incubation volume [3]. The cells were
incubated with Alamar Blue for 24 hours. The proliferation of cultures with Alamar Blue was
determined after 48h of cell culture. Every measurement was repeated two times for every well
and each treatment was performed at least three times.
2/Tkymidi~ei~coporattion.
Incorporation of [3H]thymidineby the cells was measured as described
by Duclos et al. [7]. Radiolabeled rH]thymidine (l,uCi/ml) was added to theca and granulosa cells
for the last 24h period of culture. Radioactivity was measured in a liquid scintillation counter
(LKB 1209 RACKBETA). Every measurement was repeated two times for every well and each
treatment was performed at least three times.
Experiment 3
This experiment was conducted to evaluate the synergistic action of oGH (100 ngiml) and oIGFs
(30 ngiml) on follicular steroidogenesis. Theca and granulosa cells obtained from medium follicles
were initially cultured in serum containing MI99 (5% calf serum) for 24h to allow cell attachment
to the plates. After 24h, medium was discarded and cells were cultured for an additional 48h in
MI99 supplemented with 0.1% BSA and various hormones as described in Experiment 2. Then the
conditioned medium was collected and estradiol (E) concentration was determined by RIA
(Spectra kits, Orion, Diagnica, Finland) supplied by Polatom (Swierk, Poland).
Statistical analysis
All data were expressed as means -+ SEM obtained from at least three different experiments
(n=3), each in triplicates. Significance of differences between the means were compared by analysis
of variance followed by Duncan's new multiple range test.
RESULTS
Effect of GH on IGF-I secretion by theca and granulosa cells
Table 1shows the effect of oGH (100 nglml) on IGF-I secretion by theca and granulosa cells cultured in vitro. The IGF-I was produced by both theca and granulosa cells in culture. The amount
of IGF-I produced by theca cells was 2.8 fold higher than by granulosa cells (p<0.01). Growth hormone added to the culture medium decreased IGF-I secretion by theca cells (p<0.05) but
increased IGF-I secretion by granulosa cells (p<0.05).
Table I. The effect of oGH (100 ngirnl) on IGF-I secretion by porcine theca interna and granulosa cells
(mean r SEM, n=4).
1
1
Medium IGF-I (pg/ml)
I
oGH
I
I
Theca cells
** p<0.01 designates the difference between IGF-I secretion by theca and granulosa cells
"h
Means within columns with different superscripts differ (p<0.05).
Granulosa cells
1
36
G H and IGFs regulation of porcine ovarian cells
Effect of GH, IGF-I and IGF-I1 on theca and granulosa cell proliferation
Insulin growth factor-I and IGF-I1 significantly (p<0.001) increased proliferation of porcine
granulosa and theca cells (figs. 1 and 2). This was demonstrated by the two independent assays.
In addition, both Alamar-Blue (fig. lb) and [3H]thymidineincorporation tests (fig. 2b) indicated a
small but significant decrease in basal and IGFs-stimulated granulosa cells proliferation under the
influence of GH. In the theca cells the decrease of cell proliferation measured by both tests was
observed under the basal and IGF-I condition (figs. l a and 2a). In contrast, the increase of IGF11- stimulated theca cell proliferation under the influence of G H measured by both tests (p<0.05)
was observed (figs. l a and 2a).
Theca cells
F
UGH
-
C
IGF-II
Granulosa cells
~b
I
IGF-I
C
IGF-I
IGF-II
I
Fig 1. The influence of growth hormone (oGH, 100 ng/ml) on control (C) and insulin-like growth factors
oIGF-I- (30 ngiml) and oIGF-II(30 ng/ml)-stimulated theca (a) and granulosa (b) cell proliferation measured
by Alamar Blue assay. * * * designates difference between control and IGF-stimulated cells (p<0.001); capital
letters refer to comparison between control and GH treated cells (p<0.05)
Kobdziejczyk et al.
I
Theca cells
2a
I 2b
37
C
IGF-I
IGF-II
Granulosa cells
C
IGF-I
IGF-II
Fig 2. The influence of growth hormone (GH) on control (C) and insulin-like growth factors (IGF-I and
IGF-11)-stimulated theca (a) and granulosa (b) cell proliferation measured by [3H]thymidyneincorporation.
** designates difference between control and IGF-stimulated cells (p<0.01).
Effect of GH on the basal and IGFs I and II- stimulated estradiol secretion by theca and granulosa cells
Insulin-like growth factors I and I1 increased significantly secretion of E by both investigated cell
types. In theca cells treatment with IGF-I resulted in a 2.24 fold increase and treatment with IGF-I1
caused a 6.73 fold increase in estradiol secretion compared to that of controls (fig. 3a). In granulosa cells both IGFs increased E secretion in a similar manner (fig. 3b).
38
GH and IGFs regulation of porcine ovarian cells
Ovine GH (100 ngiml) increased basal estradiol secretion by granulosa cells (2.36 vs. 3.03 ng/rnl)
but decreased IGF-I (4,58 vs. 3,81 ng/rnl)- and IGF-I1 (4.92 vs. 3.1 ng/ml)-stimulated estradiol
secretion (fig. 3b).
In contrast to granulosa cells, G H significantly decreased basal estradiol secretion in theca
cells. Additionally, in these cells the synergistic stirnulatoly effect of GH and each of IGFs on
estradiol secretion was observed (fig. 3a).
Theca cells
C
IGF-I
IGF-II
I
Granulosa cells
C
IGF-I
IGF-II
Fig 3. Effect of growth hormone (GH; 100 ngiml) on basal and insulin-like growth factors IGF-I-(30 ngiml)
and IGF-11-(30 ngiml) stimulated estradiol secretion by theca (a) and granulosa cells (b). * * designates difference between control and IGF-stimulated cells (p<0.01); capital letters refer to comparison between control
and GH treated cells ( ~ 4 0 . 0 5 ) .
Kolodziejczyk et al.
DISCUSSION
Results of the present study demonstrated the ability of porcine granulosa and theca cells to synthesise IGF-I in vbo.The exogenous GH had shown the opposite effect on IGF-I synthesis by the
two examined cell types. G H treatment decreased IGF-I secretion by theca cells but increased
IGF-I secretion by granulosa cells. Hammond ct al. [15] indicated that IGF-I is expressed in the
pig ovary and is regulated in a physiologically meaningful fashion. In addition, Hsu and Hammond
[19,20] showed that granulosa cells from 1-3 mm follicles of immature gilts secrete IGF-I in serum
free cultures for 7-10 days and it respond to growth hormone. Moreover, IGF-I1 mRNA and peptide were also detected in porcine ovary [16]. This data confirm our observation that both cell types
cultured in vitro produced IGF and that G H increased IGF-I secretion by granulosa cells.
Surprisingly these are the first data showing the influence of G H on IGF-I secretion by theca cells.
Geisthovel et al. [8], Hammond et al. [14] and Adashi et al. [2] demonstarted that insulin-like
growth factors are synthesized in ovaries of several mammalian species. Granulosa and theca cells
can synthesize IGF-I andlor IGF-11 and both cell types are equipped with receptors for IGFs. Lucy
et al. [22], however, showed that bovine and ovine granulosa cells do not release IGF-I in response
to G H in vitro.Yoshimura et al. [35] showed changes in the tissue concentrations of IGF-I in rabbit ovaries perfused with GH. These changes paralleled those triggered by exposure to hCG and
suggested that G H amplifies gonadotropin actions by stimulating IGF-I production. Sirotkin and
Makarevich [28] found that the addition of GH to the culture medium stimulated IGF-I secretion
by bovine granulosa cells collected during the early and mid-follicular phase of the estrous cycle.
Low concentration of GH stimulated estradiol output and decreased the incidence of apoptosis in
cultured cells.
In the presented study we observed a two fold increase in estradiol secretion by theca and granulosa cells under the influence of IGF-I. Zhou et al. [36] showed that IGF-I and IGF-I receptor
mRNA were selectively expressed by porcine granulosa cells, whereas IGF-I1 mRNA was found in
granulosa and theca cells suggesting that IGF-I may be more involved in the regulation of granulosa cell function than IGF-11. Presented data clearly showed high stimulatory effect of IGF-I1 on
estradiol secretion by both theca and granulosa cells.
Synergistic action of G H and IGF-I or IGF-I1 on E secretion was found to depend on the cell
type. In theca cell culture both IGFs stimulated estradiol secretion to a greater extent compared to
granulosa cells. Furthermore, their synergistic stimulatory effect on estradiol secretion was
observed in this type of cells. Theca-interna cells play a critical role in the control of ovarian function, including maintenance of structural integrity and regulation of follicular function. In particular, theca cells provide essential steroid substrates for granulosa cell steroidogenesis and modulate
granulosa function by releasing several paracrine factors. Porcine theca cells also have active P450
aram and can aromatize endogenous testosterone at autocrine manner [32]. Hernandez et al. [16]
indicated that IGF-I augmented in vipo LH- or hCG-stimulated androgen secretion in rat thecainterstitial cells. Apa et al. [4] demonstrated that GH directly stimulated androgen synthesis by rat
theca-interstitial cells since the addition of anti-IGF antibodies to the GH culture did not modify
the G H action. Previously, we have observed direct stimulatory effect of G H on estradiol secretion
by theca cells isolated from small, medium and large preovulatory follicles 1111. In the present
study we observed a decrease of estradiol secretion by theca cells under the influence of GH. This
discrepancy could be due to different culture conditions. In the previous experiment, cells were
cultured in medium supplemented with 5% of calf serum, while in the present study in serum free
medium. Gong et al. [9] using bovine granulosa cells obtained from follicles of different size
showed that recombinant bovine somatotrophin did not affect steroidogenesis by granulosa cells
from small and medium follicles cultured under serum-free condition. However, IGF-I stimulated
progesterone and estradiol secretion by cells collected from all three size categories of follicles.
40
GH and ZGFs regulation of porcine ovarian cells
The synergistic action of G H and IGFs was inhibitory in granulosa cells. Combined treatment
of G H with IGF-I or IGF-I1 resulted in a decrease of IGF-stimulated estradiol secretion by granulosa cells. Xu et aL2showed that G H is capable of amplifying IGF-I induced progesterone biosynthesis by cultured porcine granulosa cells. In this case it can stimulated granulosa cell luteinization.
It was previously shown that a high level of progesterone might function as an inhibitor of aromatase activity [12, 13, 271.
The presented data showed additionally that proliferation of both cell types was increased
under the influence of IGF-I and IGF-I1 but modestly inhibited by GH. Adashi et al. [I] suggested
that insulin and IGF-I stimulated granulosa cells mitogenesis and steroidogenesis of follicular
cells. Savion et al. [26], Li et al. 1211, Baranao & Hammond 151 showed mitogenic activity of granulosa cells induced by IGF-I without gonadotropin stimulation. Monniaux et al. [24] showed that
IGF-I stimulated proliferation in ovine granulosa cells isolated from small antral follicles and
staroidogenesis in cells from large antral ones. The observed modest inhibitory effect of G H on
cell proliferation suggests that G H can act as a stimulator of steroidogenesis but not of proliferation or survival. This is in agreement with Mondschein et al. [23] who suggested that IGFs
appeared to be more important to porcine granulosa cell cytodifferentiation than to cell replications. Moreover, they suggested that the interaction of exogenous IGFs with gonadotropins works
to inhibit replicate activity and stimulate steroidogenesis.
Thus, the present data demonstrated different action of G H on IGF-I and estradiol secretion
by granulosa and theca cells. G H alone decreased IGF-I and estradiol accumulation by theca cells
collected from medium-size follicles and cultured in serum-free medium when compared to controls. However, in the presence of IGF-I and IGF-11, G H proved to be a potent amplifier of IGFs
on estradiol production in these cells. In granulosa cells the opposite action of G H on IGF-I and
estradiol secretion was observed suggesting divergent pathways of action. In both cell types the
effect of G H on cell proliferation was rather inhibitory than stimulatory. It seems that in theca cells
G H potentiated IGF-induced cell differentiation and consequently estradiol production. In granulosa cells it stimulated cell luteinization and, as a result, progesterone secretion as shown by Xu et
al. [37]. Progesterone in turn could be responsible for inhibition synthesis of estradiol. Further
experiments are necessary to confirm this hypothesis.
ACKNOWLEDGEMENTS
The authors wish to thank Dr. M. Mika for radioimmunological determinations of steroid hormones. This work was supported by the grant BW/IZ/47/2000.
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