BIOLOGY OF REPRODUCTION 63, 1214–1218 (2000)
Growth Differentiation Factor-9 Stimulates Rat Theca-Interstitial Cell
Androgen Biosynthesis1
Elena V. Solovyeva,3 Masaru Hayashi,5 Karen Margi,3 Claudine Barkats,3 Cynthia Klein,5
Abraham Amsterdam,4 Aaron J.W. Hsueh,5 and Alex Tsafriri2,3
Bernhard Zondek Hormone Research Laboratory, Department of Biological Regulation3 and Molecular Cell Biology,4
Weizmann Institute of Science, Rehovot, 76100, Israel
Division of Reproductive Biology,5 Department of Gynecology & Obstetrics, Stanford University School of Medicine,
Stanford, California 94305-5317
ABSTRACT
Growth differentiation factor-9 (GDF-9) was shown recently
to be essential for early follicular development, including the
appearance of the theca layer. Theca cells provide the androgen
substrate for aromatization and estrogen production by granulosa cells. Using biologically active recombinant GDF-9 (rGDF9) and an androgen-producing immortalized theca-interstitial
cell (TIC) line or primary TIC, we have examined the action of
this paracrine hormone on theca cell steroidogenesis. The effect
of GDF-9 on TIC progesterone synthesis was marginal and inconsistent in the primary cultures. In immortalized theca cells,
GDF-9 attenuated the forskolin-stimulated progesterone accumulation. More significantly, this oocyte-derived growth factor
enhanced both basal and stimulated androstenedione accumulation in the primary and transformed TIC cultures. The effects
of GDF-9 on steroidogenesis by preovulatory follicles were relatively modest. Likewise, it did not affect the maturation of follicle-enclosed oocytes. The effect of GDF-9, an oocyte product,
on TIC androgen production suggests a regulatory role of the
oocyte on theca cell function and hence on follicle development
and differentiation. This direct effect of GDF-9 on thecal steroidogenesis is consistent with its recently demonstrated actions
on thecal cell recruitment and differentiation.
FSH, increased the growth of rat preantral follicles in vitro
and a-inhibin content in explants of neonatal rat ovaries
[4]. These findings suggest, therefore, that GDF-9 plays an
important role in follicular growth and differentiation. Because ovarian GDF-9 mRNA expression is limited to the
oocyte, it appears that the oocyte plays an essential regulatory role in follicle development.
Thecal cells provide structural integrity for the follicle
and are in close proximity to the basement membrane
which surrounds the mural granulosa cells. A critical intrafollicular interaction involves the provision of androgens
by the theca for aromatization in granulosa cells [7]. It was
suggested that thecal P450c17a activity and, hence, androgen
production, is regulated by paracrine factors from granulosa
cells [8, 9]. Thus, inhibin and IGF were shown to augment
LH-stimulated androgen production in rat and human thecainterstitial cells (TICs) [8–12]. Availability of recombinant
rat GDF-9, with proven biological activity [4], rat primary
TIC cultures, and an immortalized rat TIC line allowed us
to directly test the effects of this oocyte-derived paracrine
regulator on thecal androgen production. The actions of
GDF-9 on theca cells were compared with those on preovulatory follicles.
follicular development, growth factors, theca cells
MATERIALS AND METHODS
INTRODUCTION
Materials
Growth differentiation factor-9 (GDF-9) is a member of
the transforming growth factor b (TGFb) superfamily. It is
expressed mainly in the gonads [1]. In ovaries it is specifically expressed in oocytes and is first detected in primary
follicles in mouse [2], rat [3, 4], and in primordial follicles
in bovine and ovine ovaries [5]. Targeted deletion of GDF9 results in a block of follicular growth at the primary follicle stage, leading to female sterility. Follicles in such mutant mice have atypical granulosa cells and the theca layer
is absent [2]. The absence of a theca layer was confirmed
by the lack of a ring of theca cells expressing cytochrome
P450 17,20 lyase (P450c17a), LH/hCGR, and c-kit mRNA,
all of which are established theca cell markers [6]. Recently, it was demonstrated that recombinant GDF-9, like
Media were purchased from Gibco (Grand Island, NY).
Fetal calf serum (FCS), BSA, glutamine, antibiotics, and
trypsin were from Biolab (Jerusalem, Israel). Forskolin
(Fsk) was from Sigma Chemical Company (St. Louis, MO).
The progesterone and androstenedione antibodies were a
generous gift from Dr. F. Kohen, of our department (Weizmann Institute of Science, Israel), labeled steroids were
from Amersham (Boston, MA), and the gonadotropins were
from Dr. A.F. Parlow and the National Hormone and Pituitary Program of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). Recombinant rat
GDF-9 was expressed in human embryonic kidney 293T
cells, as previously described [4]. The medium contained 1
ng/ml GDF-9 activity, and conditioned medium from untransfected 293T cells was used as an additional control.
Supported by The Maria and Bernhard Zondek Hormone Research Fund.
A.T. is the incumbent of the Hermann and Lilly Schilling Foundation Professorship.
2
Correspondence. FAX: 972 8 934 4116;
e-mail: [email protected]
1
Received: 5 January 2000.
First decision: 24 February 2000.
Accepted: 2 June 2000.
Q 2000 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
Animals
Rats derived from a Wistar colony were from the Department of Hormone Research. They were provided with
water and rat chow ad libitum and housed in air-conditioned rooms that were illuminated 14 h/day. The experiments were carried out in accordance with the principles
and guidelines for the use of laboratory animals and approved by the institutional research animal committee. For
1214
GDF-9 STIMULATES THECA-INTERSTITIAL CELL ANDROGEN
culture of preovulatory follicles, immature rats were injected with eCG (10 IU) between 0900 and 0930 h on Days
23–24 of age to enhance multiple follicular development.
TIC Primary Cultures and Their Immortalization
Immature 23- to 24-day-old rats were anesthetized and
hypophysectomized by an intra-aural approach. TICs were
isolated 4–5 days after hypophysectomy essentially according the method described by Magoffin [13]. The primary
TIC cultures responded to LH or Fsk stimulation with androgen and progesterone production, but FSH had no effect
and estrogen could not be detected in the culture medium,
in accordance with previously published data [14]. Primary
TICs were cultured (25 3 103 cells/well) in 24-well culture
plates (Falcon, Meylan Cedex, France) in 1 ml of Hepesbuffered medium 199 with 5% FCS for 24 h, followed by
24–48 h in serum-free medium containing 0.1% BSA with
the indicated doses of GDF-9 or the untransfected 293T
cell conditioned medium (CM) with and without LH (100
ng/ml) or Fsk (100 mM). The media were frozen and saved
for steroid radioimmunoassay (RIA).
TIC cell lines were obtained as described previously for
rat granulosa cells [15] by triple transfection of primary
TIC cultures with the following plasmids: pSVBam containing the entire SV40 genome, pEJ 6.6. encoding activated human Ha-ras oncogene, and pSV-LH/hCG-R, containing the complete coding region of LH receptor cDNA.
The cells were cultured (25–50 3 104) in 100-mm Petri
dishes (Falcon) with 10 ml Hepes-buffered medium 199
containing 5% FCS for 48 h and transfected with 5 mg
pSVBam, 5 mg pEJ 6.6, and 5 mg of pSV-LH/hCG-R by
the calcium phosphate procedure [16]. The coprecipitate
was allowed to remain on the cells for 5 h. The medium
was changed every 3–4 days. Densely growing foci of immortalized cells were visualized and selected after 3 wk
and transferred to 24-well culture plates. After 2–3 days,
stably growing cells were transferred to larger dishes. When
approaching confluence, the cells were collected in freezing
vials and kept in liquid nitrogen.
Reverse Transcriptase-Polymerase Chain
Reaction Analysis
The expression of P450c17a was examined by relativequantitative reverse transcriptase-polymerase chain reaction
(RT-PCR) as previously described [17]. Total RNA was extracted from immortalized or primary TICs and from preovulatory follicles by the acid-guanidium-phenol-chloroform
method [18] and aliquots of 1.8 mg RNA from transformed
TICs or 200 ng RNA from primary TICs or preovulatory
follicles were reverse-transcribed using random primers followed by PCR amplification. RT reactions contained 50 units
of MMLV-RT, 200 mM dNTP, 6.5 mM MgCl2, 20 units of
RNAsin, 500 mg oligo(dT), and 1.53 PCR buffer (Promega). The reaction was performed at 378C for 2 h. Fragments
of the reverse-transcribed P450c17a cDNA were amplified using a labeled nucleotide ([a-32P]dCTP, Amersham) and the
following pairs of primers were employed: 59GTCACTGTGTGATATGATGCTGGC-39 and 59-GTTCAGGCATGAACTGATCTGGCT-39 [19]. A fragment of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) cDNA
that had served as an internal standard was amplified in parallel using the primers 59-GCCATCAACGACCCCTTCAT39 and 59-TTCACACCCATCACAAACAT-39.
PCRs were further performed in the same RT test tube
that contained 250 ng of each primer, 200 mM dNTP, 2.5
1215
mM MgCl2, 2 mCi [a-32P]dCTP, 13 PCR buffer (Promega), and 2.5 units Taq polymerase. Annealing was performed at 658 and 558C during 33 and 22 cycles for
P450c17a and GAPDH, respectively. The RNA input and
the number of cycles for P450c17a and GAPDH were selected within the linear range. The radioactive products
were electrophoresed on 5% nondenaturing polyacrylamide
gel in 0.53 Tris-Boric acid-EDTA buffer. Gels were dried,
and radioactivity was determined by exposure to x-ray film
for 1 h at 2708C. Quantitation and comparison of the autoradiograms were performed by densitometric analysis
(Quantity One, PDI, 420oe, New York, NY) and the expression level normalized to GAPDH.
Effect of GDF-9 on Immortalized Theca
Cell Steroidogenesis
Following fast thawing and removal of Dulbecco modified Eagle medium, the cells were plated onto 100-mm
Falcon culture dishes in medium 199 with 10% FCS for
the first 48 h and 5% FCS afterward, and cultured to subconfluence. For assays, the cells (25 3 103) were plated
onto 24-well culture plates (Falcon) with 5% serum for 24
h. Afterward, the medium was replaced with serum-free
medium containing 0.1% BSA with the indicated doses of
GDF-9 [4] or the untransfected 293T cell CM with and
without Fsk (100 mM) in quadruplicates and cultured for
24 h. The media were collected and saved for RIA at
2708C.
Follicle Cultures and Examination of Oocytes
Immature Wistar-derived rats treated with eCG (10 IU)
were killed on the morning of the day of proestrus by cervical dislocation. Preovulatory follicles were excised under
a dissection microscope as previously described [20]. The
follicles, 5–10 per dish, were cultured for 8 or 24 h in
Leibovitz’s L-15 medium supplemented with 0.1% BSA
alone or with LH (1 mg/ml) or with a combination of LH
and the indicated concentration of GDF-9.
At the end of the 8-h culture period, follicle-enclosed
oocytes were released by making a small incision in the
follicle, and examined for meiotic status by Nomarski interference microscopy (Zeiss, Oberkochen, Germany). Oocytes showing a clear nuclear membrane (germinal vesicle)
or intact nucleolus only were classified as immature. Oocytes that did not show any nuclear structures because they
had undergone germinal vesicle breakdown were classified
as mature [20].
Radioimmunoassay and Statistics
Progesterone and androstenedione accumulated during
the indicated culture period was determined by previously
established RIA [21, 22]. In our initial cultures we compared the accumulation of androstenedione and a-reduced
androsterone using an antiserum that had been kindly provided by Drs. G. Barbe and D.T. Armstrong of the University of Western Ontario. These two androgens were within
the same range (data not shown). Therefore, only androstenedione was assayed in all the experiments. Statistical
differences between groups were analyzed by Students ttest and ANOVA. P , 0.05 was considered significant.
RESULTS
Immortalized TICs
In immortalized TICs that had been cultured for 24 h in
a serum-free medium, GDF-9 dose-dependently increased
1216
SOLOVYEVA ET AL.
FIG. 1. The effect of GDF-9 on steroid accumulation by an immortalized
TIC line. A) Androstenedione (A4). B) Progesterone (P4). UC, Untreated
control; CM, untransfected 293T cell conditioned medium; Fsk, forskolin,
100 mM. The number of replicate cultures is indicated on the columns.
Means with different letter superscripts differ from their corresponding
treatment control (b, P , 0.05; c, P , 0.005).
androstenedione accumulation both in cultures with and
without Fsk, while it decreased Fsk-stimulated theca cell
progesterone accumulation. The data of each experiment
were normalized as a percentage of the untreated control
and the results of at least three different experiments were
recorded (Fig. 1). GDF-9 stimulated TIC androgen accumulation from 8.8 6 0.4 to 27.8 6 0.7 pg/ml per 24 h21
at the 10 and 500 ng/ml doses, respectively. The stimulation
reached significance from 100 ng/ml GDF-9 vs. the control
and from 250 ng/ml vs. the CM control. When GDF-9 was
added to Fsk-stimulated cultures it further enhanced androgen production from 21.5 6 1.7 to 44.7 6 5.6 pg/ml per
24 h21 at 10 and 500 ng/ml (Fig. 1A). By contrast, progesterone accumulation (Fig. 1B) was not affected by GDF9 in control cultures (20–30 pg/ml per 24 h21). Forskolin
stimulated progesterone synthesis approximately 10-fold,
but the addition of GDF-9 to Fsk-treated cultures suppressed progesterone synthesis in a dose-dependent manner
from 239.9 6 27.4 to 26.3 6 6.8 pg/ml per 24 h21 at doses
of 10 and 500 ng/ml, respectively.
FIG. 2. The effect of GDF-9 on steroid accumulation by primary rat TIC
during 24-h culture. LH: 100 ng/ml. Other details as in Figure 1.
9 had no consistent effect on TIC progesterone production
when added to LH- or Fsk-treated cultures (Fig. 2B). Extension of the culture to 48 h did not change this general
pattern of steroidogenesis of primary TIC cultures (data not
shown).
Expression of P450c17a
Relative-quantitative RT-PCR confirmed the expression
of P450c17a mRNA in primary (data not shown) and transformed TICs and in preovulatory follicles (Fig. 3). In the
follicle and primary TIC, only one band of P450c17a mRNA
Primary TIC Cultures
Androstenedione and progesterone production by primary TICs that had been cultured for 24 h in serum-free
medium is summarized in Figure 2 as a percentage of the
untreated control in each experiment and included at least
two separate experiments. GDF-9 stimulated accumulation
of androstenedione (from 36 6 4 in the control to 77 6 6
pg/ml per 24 h21 at the 250 ng/ml dose) and progesterone
(from 0.35 6 0.01 to 1.10 6 0.08 ng/ml per 24 h21 at the
250 ng/ml dose) in a dose-dependent manner. Addition of
LH or Fsk increased androstenedione accumulation compared with the control. GDF-9 alone or in combination with
LH or Fsk stimulated androstenedione in a dose-dependent
manner, reaching significance at the highest dose (Fig. 2A).
LH and Fsk stimulated TIC progesterone synthesis. GDF-
FIG. 3. RT-PCR analysis of expression of P450c17a mRNA in immortalized TICs. On the far right lane is the P450c17a mRNA band in preovulatory
follicles explanted 48 h after eCG treatment of immature rats. The five
left lanes show the three bands obtained from immortalized TICs. Competition with inner primer greatly reduced only the upper band, confirming this is the P450c17a mRNA band.
GDF-9 STIMULATES THECA-INTERSTITIAL CELL ANDROGEN
TABLE 1. Steroidogenesis in rat preovulatory follicles and GDF-9. Each
of the replicate cultures included 5 follicles.
Treatment
Control
CM (ml/ml)
200
600
GDF-9 (ml/ml)
60
200
600
LH (1 mg/ml)
LH (1mg/ml)
1CM (ml/ml)
200
600
LH (1mg/ml)
1GDF-9 (ml/ml)
20
60
200
600
Number
of
Replicates
P4
(ng/foll/8 h21)
Mean6SEM
A4
(ng/foll/8 h21)
Mean6SEM
12
0.16 6 0.02
0.51 6 0.03
8
3
0.16 6 0.01
0.17 6 0.20
0.44 6 0.05
0.49 6 0.06
3
8
3
11
9
3
9
8
11
7
0.21
0.22
0.30
3.71
6
6
6
6
0.03
0.03
0.03
0.41
3.80 6 0.30
3.85 6 0.23
5.90
4.80
5.63
3.80
6
6
6
6
0.82**
0.94
0.86**
0.45
0.40
0.52
0.57
3.71
6
6
6
6
0.05
0.03
0.07
0.29
3.80 6 0.24
4.84 6 0.13*
4.15
4.35
3.33
3.91
6
6
6
6
0.28
0.36*
0.14
0.46
*P , 0.05; **P , 0.01.
was seen. The transformed TICs showed three close bands
on RT-PCR (Fig. 3), of which only the upper one was reduced by competition with an inner primer within the same
fragment. Therefore, only the upper band was taken as
P450c17a. In transformed cells, treatment with Fsk and
GDF-9 combined resulted in a higher (3-fold) increase in
P450c17a mRNA expression, while each one of these treatments separately was less effective (1.6-fold stimulation)
compared with the untreated controls.
Preovulatory Follicle Cultures
The effects of GDF-9 on preovulatory follicle steroid
accumulation differed from those on TIC cultures. GDF-9
did not significantly affect follicular steroidogenesis in control cultures without LH. The effect on progesterone accumulation was biphasic. GDF-9 augmented (20–200 ng/
ml) progesterone stimulation by LH, but at the highest concentration tested (600 ng/ml), it was without effect. In contrast to theca cell cultures, androgen accumulation in the
whole follicle was not markedly affected by GDF-9 (Table
1). Likewise, GDF-9 (20–200 ml/ml) did not affect the maturation of follicle-enclosed oocytes during an 8-h culture.
Follicle-enclosed oocytes cultured without LH remained
immature (with intact germinal vesicle) and those stimulated with LH resumed meiosis, irrespective of the presence
of GDF-9 (data not shown).
DISCUSSION
The oocyte-derived growth factor, GDF-9, enhanced
both basal and stimulated androstenedione accumulation in
primary and transformed TIC cultures. This effect of GDF9 on TIC cells was consistent, despite the low responsiveness of the cells to LH in some of the experiments. Another
difficulty encountered in these experiments was the tendency of the untransfected 293T cell CM to increase TIC androstenedione accumulation over the control. Nevertheless,
the comparable doses of GDF-9 showed a higher increase
in androgen accumulation than CM (Fig. 1A, untreated and
Fsk-treated, P , 0.05; Fig. 2A, untreated and Fsk-treated
P , 0.0001; LH-treated, P , 0.01). While the reason for
1217
increased steroidogenesis in CM vs. control remains to be
determined, collectively, the data show a direct effect of
GDF-9 on TIC androgen production.
The effect of GDF-9 on TIC progesterone synthesis was
marginal and inconsistent in primary cultures. In immortalized theca cells, GDF-9 attenuated the Fsk-stimulated
progesterone accumulation. Likewise, the effects of GDF9 on preovulatory follicles were relatively modest. The
stimulation of follicular progesterone accumulation probably reflects the action of GDF-9 on granulosa cell steroidogenesis. Indeed, it has been shown recently that GDF-9
stimulates granulosa cell steroidogenic acute regulator protein (StAR) mRNA and progesterone synthesis [23]. Vitt et
al. [24] have recently demonstrated GDF-9 stimulation of
basal steroidogenesis in granulosa cells from small antral
and preovulatory follicles, but suppression of FSH-stimulated steroidogenesis.
Recently, the role of GDF-9 and, therefore, of the oocyte, in the initiation of primordial follicle growth [2] and
in the continued growth and differentiation of early follicles
[4] have been demonstrated. Furthermore, theca cells were
absent in the aberrant follicles of GDF-9-targeted mutant
mice [2], suggesting that the factor is either directly involved in the recruitment and differentiation of theca cells
or acts indirectly on theca development through other ovarian cells. The present data provide evidence for a direct
stimulatory action of GDF-9 on theca cell androgen synthesis and suggest the presence of its specific receptors in
primary TIC cultures as well as in immortalized cells. The
observed discrepancy in the ability of GDF-9 to stimulate
TIC androstenedione production and the lack of a clear-cut
stimulatory effect on androgen (and estrogen, data not
shown) synthesis in preovulatory follicles is probably related to the stage of theca cell differentiation. Penultimate
follicular growth is associated with increased expression of
the TIC androgen-producing P450c17a, whereas in preovulatory follicles, P450c17a reaches its peak [25] and this activity decreases after LH/hCG stimulation [26]. Furthermore, the higher GDF-9 expression in oocytes of primary
and large antral rat follicles [3, 4] suggests that GDF-9 is
more abundant and active during earlier stages of follicular
development. Alternatively, the reduced sensitivity of the
preovulatory follicle to GDF-9 in terms of androgen synthesis may be related to other paracrine interactions that
attenuate this GDF-9 action.
Thecal androgen synthesis is a necessary requirement in
later stages of follicular development [7, 14] and several
paracrine systems were suggested to mediate the stimulatory actions of FSH on granulosa cells to increase theca
cell androgen production. Thus, IGF-1 and inhibin augmented the LH-stimulation of androgen synthesis in rat and
human theca cells in vitro [8–12]. Using primary cultures
of neonatal rat granulosa cells, Magoffin and his colleagues
[27, 28] presented evidence for an undefined theca-cell differentiation factor (TDF) from preantral follicles, which
stimulated androgen production and the expression of LH
receptor and steroidogenic enzymes. Given the early expression of GDF-9 in the oocyte, it is possible that these
effects ascribed to TDF are the result of GDF-9 or similar
oocyte-derived paracrine hormones. Additional paracrine
hormones similar to GDF-9, such as bone morphogenetic
protein 15 (BMP-15/GDF-9B) and BMP-6/Vgr-1 are expressed in oocytes at an early stage of follicular development [29–32].
Most recently, it has been shown that GDF-9 also affects
later stages of follicular development, including stimulation
1218
SOLOVYEVA ET AL.
of granulosa cell hyaluronan synthase 2, cyclooxygenase 2,
and StAR mRNA expression, and suppression of urokinase
and LH/hCG receptor mRNA synthesis [23]. Some of these
participate not only in follicular growth, but also in the
ovulatory response. Likewise, GDF-9 was shown to enhance preantral follicle growth [4] and granulosa cell proliferation, but it attenuated FSH-stimulated steroidogenesis
and LH receptors [24]. These effects were seen in granulosa
cells from both preantral and preovulatory follicles.
In conclusion, the stimulation of TIC androgen production in vitro by GDF-9, demonstrated here, suggests a role
for an oocyte-derived hormone in the regulation of theca
cell steroidogenesis. Thus, GDF-9 seems to directly affect
both somatic cellular compartments of the follicle, granulosa and theca cells. Because reciprocal effects of theca on
oocyte meiosis have also been suggested recently [33], intrafollicular interactions between the oocyte and theca cells
seem to be important for follicular development and function.
ACKNOWLEDGMENTS
We thank M. Popliker, A. Dantes, and A. Tsafriri for their excellent
technical help and Dr. A.F. Parlow and the NIDDK National Hormone &
Pituitary Program, National Institute of Child Health and Human Development for gonadotropins.
13.
14.
15.
16.
17.
18.
19.
20.
21.
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