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ARTICLES |
a Bernhard Zondek Hormone Research Laboratory, Department of Biological Regulation and
b Molecular Cell Biology, Weizmann Institute of Science, Rehovot, 76100, Israel
c Division of Reproductive Biology, Department of Gynecology & Obstetrics, Stanford University School of Medicine, Stanford, California 94305-5317
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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follicular development, growth factors, theca cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), LH/hCGR, and c-kit mRNA, all of which are established theca cell markers [6]. Recently, it was demonstrated that recombinant GDF-9, like FSH, increased the growth of rat preantral follicles in vitro and -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 P450c17 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 theca-interstitial 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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/µl GDF-9 activity, and conditioned medium from untransfected 293T cells was used as an additional control.
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 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 x 103 cells/well) in 24-well culture plates (Falcon, Meylan Cedex, France) in 1 ml of Hepes-buffered 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 µM). 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 x 104) in 100-mm Petri dishes (Falcon) with 10 ml Hepes-buffered medium 199 containing 5% FCS for 48 h and transfected with 5 µg pSVBam, 5 µg pEJ 6.6, and 5 µg 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 P450c17 was examined by relative-quantitative 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 µg 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 1.5x PCR buffer (Promega). The reaction was performed at 37°C for 2 h. Fragments of the reverse-transcribed P450c17 cDNA were amplified using a labeled nucleotide ([-32P]dCTP, Amersham) and the following pairs of primers were employed: 5'-GTCACTGTGTGATATGATGCTGGC-3' and 5'-GTTCAGGCATGAACTGATCTGGCT-3' [19]. A fragment of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) cDNA that had served as an internal standard was amplified in parallel using the primers 5'-GCCATCAACGACCCCTTCAT-3' and 5'-TTCACACCCATCACAAACAT-3'.
PCRs were further performed in the same RT test tube that contained 250 ng of each primer, 200 mM dNTP, 2.5 mM MgCl2, 2 mCi [-32P]dCTP, 1x PCR buffer (Promega), and 2.5 units Taq polymerase. Annealing was performed at 65° and 55°C during 33 and 22 cycles for P450c17 and GAPDH, respectively. The RNA input and the number of cycles for P450c17 and GAPDH were selected within the linear range. The radioactive products were electrophoresed on 5% nondenaturing polyacrylamide gel in 0.5x Tris-Boric acid-EDTA buffer. Gels were dried, and radioactivity was determined by exposure to x-ray film for 1 h at -70°C. 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 x 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 µM) in quadruplicates and cultured for 24 h. The media were collected and saved for RIA at -70°C.
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 µg/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 -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 t-test and ANOVA. P < 0.05 was considered significant.
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RESULTS |
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In immortalized TICs that had been cultured for 24 h in a serum-free medium, GDF-9 dose-dependently increased 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 ± 0.4 to 27.8 ± 0.7 pg/ml per 24 h-1 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 ± 1.7 to 44.7 ± 5.6 pg/ml per 24 h-1 at 10 and 500 ng/ml (Fig. 1A). By contrast, progesterone accumulation (Fig. 1B) was not affected by GDF-9 in control cultures (20–30 pg/ml per 24 h-1). 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 ± 27.4 to 26.3 ± 6.8 pg/ml per 24 h-1 at doses of 10 and 500 ng/ml, respectively.
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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 ± 4 in the control to 77 ± 6 pg/ml per 24 h-1 at the 250 ng/ml dose) and progesterone (from 0.35 ± 0.01 to 1.10 ± 0.08 ng/ml per 24 h-1 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-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).
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Expression of P450c17
Relative-quantitative RT-PCR confirmed the expression of P450c17 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 P450c17 mRNA 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 P450c17. In transformed cells, treatment with Fsk and GDF-9 combined resulted in a higher (3-fold) increase in P450c17 mRNA expression, while each one of these treatments separately was less effective (1.6-fold stimulation) compared with the untreated controls.
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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 µl/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).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 GDF-9 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 P450c17, whereas in preovulatory follicles, P450c17 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 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 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; alex.tsafriri{at}weizmann.ac.il
Accepted: June 2, 2000.
Received: January 5, 2000.
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REFERENCES |
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