Growth Differentiation Factor-9 Stimulates Rat Theca-Interstitial Cell Androgen Biosynthesis

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 (rGDF-9) 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. Introduction Growth differentiation factor-9 (GDF-9) is a member of the transforming growth factor β (TGFβ) 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 GDF-9 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 (P450c17α), 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 α-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 P450c17α 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. Materials and Methods Materials 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 × 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 × 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 P450c17α was examined by relative-quantitative reverse transcriptase-polymerase chain reaction (RT-P (...truncated)