329 Growth differentiation factor-9 has divergent effects on proliferation and steroidogenesis of bovine granulosa cells

L J Spicer, P Y Aad, D Allen, S Mazerbourg1 and A J Hsueh1 Department of Animal Science, Oklahoma State University, Stillwater, Oklahoma 74078, USA 1Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California 94305-5317, USA (Requests for offprints should be addressed to L J Spicer; Email: [email protected])

Abstract In addition to gonadotropins, steroidogenesis and prolifer- 600 ng/ml GDF-9 decreased (P<0·05) progesterone pro- ation of granulosa cells during follicular development are duction (by 28%), increased (P<0·05) cell numbers (by controlled by a number of intraovarian factors including 60%), and had no effect (P>0·10) on estradiol production. growth differentiation factor-9 (GDF-9), bone morpho- In FSH plus IGF-I-treated granulosa cells obtained from genetic protein-4 (BMP-4), and IGF-I. The objective of large follicles, GDF-9 caused a dose-dependent decrease this study was to determine the effect of GDF-9 and (P<0·05) in IGF-I-induced progesterone (by 13–48%) BMP-4 and their interaction with IGF-I and FSH on and estradiol (by 20–51%) production. In contrast, GDF-9 ovarian granulosa cell function in cattle. Granulosa cells increased basal and IGF-I-induced granulosa cell numbers from small (1–5 mm) and large (8–22 mm) follicles were by over 2-fold. Furthermore, treatment with BMP-4 also collected from bovine ovaries and cultured for 48 h in inhibited (P<0·05) steroidogenesis by 27–42% but had no medium containing 10% fetal calf serum and then treated effect on cell numbers. To elucidate downstream signaling with various hormones in serum-free medium for an pathways, granulosa cells from small follicles were trans- additional 48 h. We evaluated the effects of GDF-9 fected with similar to mothers against decapentaplegics (150–600 ng/ml) and BMP-4 (30 ng/ml) during a 2-day (Smad) binding element (CAGA)- or BMP response exposure on hormone-induced steroidogenesis and cell element (BRE)-promoter reporter constructs. Treatment proliferation. In FSH plus IGF-I-treated granulosa cells with GDF-9 (but not BMP-4) activated the Smad3- obtained from small follicles, 300 ng/ml GDF-9 reduced induced CAGA promoter activity, whereas BMP-4 (but (P<0·05) progesterone production by 15% and 600 ng/ml not GDF-9) activated the Smad1/5/8-induced BRE pro- GDF-9 completely blocked (P<0·01) the IGF-I-induced moter activity. We have concluded that bovine granulosa increase in progesterone production. In comparison, 300 cells are targets of both GDF-9 and BMP-4, and that and 600 ng/ml GDF-9 decreased (P<0·05) estradiol pro- oocyte-derived GDF-9 may simultaneously promote duction by 27% and 71% respectively, whereas 150 ng/ml granulosa cell proliferation and prevent premature GDF-9 was without effect (P>0·10). Treatment with differentiation of the granulosa cells during growth of 600 ng/ml GDF-9 increased (P<0·05) numbers (by 28%) follicles, whereas theca-derived BMP-4 may also prevent of granulosa cells from small follicles. In the same cells premature follicular differentiation. treated with FSH but not IGF-I, co-treatment with Journal of Endocrinology (2006) 189, 329–339

Introduction Mazerbourg & Hsueh 2003, Juengel & McNatty 2005). Expression of two of these TGF-
family members, During ovarian follicular development, granulosa cell GDF-9 and BMP-15, is confined to the oocyte of primary proliferation and differentiation are influenced by the and larger follicles in cattle (Sendai et al. 2001, Lonergan gonadotropins and different intraovarian factors secreted et al. 2003, Pennetier et al. 2004), sheep (Bodensteiner by both the oocyte and the surrounding somatic cells et al. 2000, Juengel et al. 2002), pigs (Prochazka et al. (McGee & Hsueh 2000, Hunter et al. 2004, Spicer 2004, 2004), and rodents (McGrath et al. 1995, Jaatinen et al. Juengel & McNatty 2005). Among the intraovarian factors 1999), whereas other BMPs such as BMP-2 and -4 are are members of the transforming growth factor (TGF)-
expressed in granulosa and/or theca cells but not the superfamily, including TGF-
, activins, bone morpho- oocyte (Erickson & Shimasaki 2003, Glister et al. 2004, genetic proteins (BMPs), and growth differentiation fac- Fatehi et al. 2005). Studies using mice (Dong et al. 1996, tors (GDFs) (Shimasaki et al. 1999, Knight & Glister 2003, Carabatsos et al. 1998) and sheep (Hanrahan et al. 2004)

Journal of Endocrinology (2006) 189, 329–339 DOI: 10.1677/joe.1.06503 0022–0795/06/0189–329  2006 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org

Downloaded from Bioscientifica.com at 09/27/2021 08:08:57AM via free access 330 L J SPICER and others · Effects of GDF-9 in bovine granulosa cells

with inactivating mutations of the GDF-9 gene have small follicles (Stewart et al. 1996, Spicer et al. 2001), and revealed an important role of this oocyte factor in the bovine follicles destined to ovulate average 102mm stimulation of early follicular growth and fertility. surface diameter (Marion et al. 1968). Granulosa cells were However, little is known about the role of GDF-9 and re-suspended in serum-free medium containing colla- BMPs in regulating granulosa cell function in single- genase and DNase (Sigma Chemical Co) at 1·25 mg/ml ovulating species such as cattle and humans. Previous and 0·5 mg/ml respectively, to prevent cell clumping studies have revealed direct effects of GDF-9 on granulosa prior to plating. Cells were maintained in this collagenase– cell functions in rats (Vitt et al. 2002, Kaivo-Oja et al. DNase-containing medium for less than 1 h prior to 2003, Roh et al. 2003). Because insulin-like growth dispersion in 1 ml 10% fetal calf serum (FCS) medium factor-I (IGF-I) is a major trophic hormone involved in without proteases. follicular development (Mazerbourg et al. 2003, Spicer Approximately 2·0105 viable cells (in 20–50 µl 2004, Hunter et al. 2004), our first objective was to collagenase–DNase medium) were plated on 24-well determine the effects of GDF-9 and BMP-4 on IGF-I- Falcon multiwell plates (Becton Dickinson, Lincoln Park, induced cell proliferation and steroidogenesis in cultured NJ, USA) in a mixture of 1:1 Dulbecco’s modified Eagle’s bovine granulosa cells. medium and Ham’s F-12 containing 10% FCS, 0·12 mM GDF-9 is most closely related to GDF-9B/BMP-15, gentamycin, 2·0 mM glutamine, and 38·5 mM sodium both having homology closer to BMPs than to the activin bicarbonate (all obtained from Sigma Chemical Co) as and TGF-
proteins (Newfeld et al. 1999, Vitt et al. 2002). previously described (Langhout et al. 1991, Spicer & Members of the TGF-
family initiate signaling by Chamberlain 1998, Spicer et al. 2002). Viability of granu- assembling type I and type II serine/threonine kinase losa cells from small and large follicles was determined by receptor complexes that activate one or more of the the trypan blue exclusion method, and averaged 5811% eight specific similar to mothers against decapentaplegic and 7912% respectively. Cells were cultured in an  (Smad) transcription factors (Massague 1998, Mazerbourg environment of 95% air and 5% CO2 at 38·5 Cin10% & Hsueh 2003). The type I receptors are also designated FCS for the first 48 h with a medium change at 24 h. Cells as activin receptor-like kinases (ALKs) and are responsible, were then washed twice with serum-free medium and the in part, for transmitting ligand specificity within target various treatments (see below) applied in serum-free cells (Massague 1998, Lux et al. 1999). GDF-9 interacts medium for 48 h with a medium change at 24 h. After the with the BMP type II receptor (BMPRII) and the type I second 24-h treatment period, medium was collected for receptor ALK5 and activates Smad2/3 (Vitt et al. 2002, steroid RIA and cells were collected for cell enumeration Kaivo-Oja et al. 2003, Roh et al. 2003, Mazerbourg (see below). et al. 2004), whereas BMP-2 and BMP-4 interact with The hormones used in the cell culture were: FSH BMPRII and the type I receptors, ALK3 and ALK6 (ovine F1913; FSH activity, 15NIH-FSH-S1 U/mg) which activate Smad1/5/8 (Massague 1998, Kawabata from Scripps Laboratories (San Diego, CA, USA), recom- et al. 1998). However, little is know about the intracellular binant human IGF-I and BMP-4 from R&D Systems signaling system GDF-9 and BMP-4 in single-ovulating (Minneapolis, MN, USA), and recombinant rat GDF-9 species such as cattle or humans. Thus, our second objec- was generated and characterized as previously described tive was to determine the intracellular signaling pathway (Hayashi et al. 1999, Vitt et al. 2000a). Briefly, expression used by GDF-9 and BMP-4 in bovine granulosa cells. vectors for wild-type and epitope-tagged GDF-9 were constructed using pcDNA3·1 Zeo (Invitrogen). N-tagged GDF-9 encoded a Flag epitope (DYKDDDDK) for the Materials and Methods M1 antibody followed by six histidine residues fused to the amino terminus of mature GDF-9. Clonal human Biological materials embryonic kidney 293T cell lines stably expressing wild- Ovaries from non-pregnant beef cows were collected type and tagged GDF-9 were used. Quantitation of from a local slaughterhouse and, based on surface diam- N-tagged GDF-9 was done after purification with a nickel eter, follicular fluid from small (1–5 mm) and large follicles column and measurement of protein content using Micro (8–22 mm) was aspirated using 20 gauge needles and 3 ml BCA protein assay kit (Perstorp Life Science, Rockford, syringes and centrifuged at 200 g for 5 min to isolate IL, USA). Purified N-tagged GDF-9 was then used as a granulosa cells as previously described (Langhout et al. standard for the quantitation of wild-type GDF-9 in the 1991, Spicer & Chamberlain 1998, Spicer et al. 2002). conditioned medium (serum free) of 293T cells by This size classification was based on previous studies immunoblots using specific GDF-9 antibodies. In experi- showing that granulosa cells from small follicles are less ments 1, 2, 3, and 5 this GDF-9 preparation was added at responsive to follicle-stimulating hormone (FSH) and 2 µl or less per ml serum-free medium (depending on dose IGF-I than are cells from large follicles (Spicer & tested). Previous studies (Vitt et al. 2000a, Roh et al. 2003) Chamberlain 1998, Spicer et al. 2002), follicles 8 mm and have shown that conditioned medium from non- greater have much larger estradiol concentrations than transfected human embryonic kidney 293T cells as well as

Journal of Endocrinology (2006) 189, 329–339 www.endocrinology-journals.org

Downloaded from Bioscientifica.com at 09/27/2021 08:08:57AM via free access Effects of GDF-9 in bovine granulosa cells · L J SPICER and others 331 medium with an inactive recombinant N-Tag GDF-9 has mones were selected based on the results of experiment 1 no effect on several granulosa cell functions evaluated. and previous in vitro studies (Vitt et al. 2000a, 2002, Mazerbourg et al. 2004) indicating that these doses are effective in altering steroidogenesis and/or promoter Experimental design activity in granulosa cells. Experiment 6 was designed to evaluate the effect Experiments 1 and 2 were designed to evaluate the of purified recombinant human GDF-9 (rhGDF-9; dose–response effect of GDF-9 on proliferation and PeproTech Inc, Rock Hill, NJ, USA) and conditioned steroidogenesis of small- and large-follicle granulosa cells medium (serum free) from non-transfected human respectively. Cells were cultured for 48 h in 10% FCS, embryonic kidney 293T cells on proliferation and washed twice with serum-free medium as described steroidogenesis of small-follicle granulosa cells. Cells were earlier, and 0, 150, 300, and 600 ng/ml GDF-9 were cultured for 48 h in 10% FCS, washed twice with applied for 48 h in the presence of either FSH (30 ng/ml) serum-free medium as described earlier, and 0 and or FSH plus IGF-I (30 ng/ml). The doses of FSH and 600 ng/ml rhGDF-9, 2 µl conditioned medium from IGF-I were selected based on previous studies indicat- non-transfected 293T cells, and/or 0 or 30 ng/ml FSH ing that these doses are maximal for stimulation of were applied for 48 h in the presence of IGF-I (30 ng/ml). estradiol secretion and/or cell proliferation (Spicer & Francisco 1997, Spicer & Chamberlain 1998, Spicer et al. 2002). The doses of GDF-9 were selected based Transfection of granulosa cells on previous studies (Vitt et al. 2000a, 2002, McNatty Granulosa cells (2105 viable cells/well) were cultured in et al. 2005a, 2005b). 24-well plates in medium supplemented with 10% FCS Experiment 3 was designed to evaluate the effect of for 2 days as described earlier. After medium change, cells GDF-9 on FCS-induced cell proliferation of large-follicle were incubated in the serum-free medium and trans- granulosa cells. Cells were cultured for 48 h in 10% FCS, fected with 250 ng DNA/well using Lipofectamine 2000 washed twice with serum-free medium as described (Invitrogen) as previously described (Mazerbourg et al. earlier, and 0, 1%, and 2% of FCS was applied for 48 h in 2004). Briefly, the pCMV-
-galactosidase plasmid (50 ng) the absence or presence of GDF-9 (300 ng/ml). The dose was co-transfected to monitor transfection efficiency. of GDF-9 was selected based on the results of experiments After transfection, cells were treated with GDF-9 or 1 and 2. BMP-4 for 24 h in medium containing 1% FCS. To Experiment 4 was designed to evaluate the effect of harvest cells, lysis buffer (200 µl; Promega Corp) was BMP-4 on proliferation and steroidogenesis of small- and added into each well and 30 µl of the supernatant was large-follicle granulosa cells respectively. Cells were cul- used for luciferase determination using a luminometer tured for 48 h in 10% FCS, washed twice with serum-free (Luminark microplate reader; Bio-Rad Laboratories, medium as described earlier, and 0 and 30 ng/ml BMP-4 Inc.). Fifty microliters of the cell lysate were also used to were applied for 48 h in the presence of FSH plus IGF-I measure the
-galactosidase activity to monitor trans- (30 ng/ml). The dose of BMP-4 was selected based on fection efficiency. The reporter activity is expressed as the previous studies indicating that this dose is maximal for its ratio of relative light units/
-galactosidase activity. effect on steroidogenesis (Shimasaki et al. 1999, Mulsant et al. 2001, Fabre et al. 2003, Sudo et al. 2004). Experiment 5 was designed to elucidate the specificity Determination of cell numbers and steroid concentrations of the GDF-9 and BMP-4 signaling pathway and, based on findings using rat granulosa cells, we tested the ability Medium was collected from individual wells and frozen at of GDF-9 and BMP-4 to stimulate the Smad binding 20 C for subsequent hormone analyses. Cells were then element (CAGA) and the BMP response element (BRE) gently washed twice with 0·9% saline (500 µl), exposed to promoter respectively in bovine granulosa cells. The 500 µl trypsin solution (0·25%, w/v) for 20 min at 25 C, CAGA and BRE promoters are known to be activated by and then scraped from each well. Cell aggregates were GDF-9 mediated by Smad3 and several BMPs mediated minimized by pipetting cell suspensions back and forth by Smad1/5/8 respectively (Dennler et al. 1998, Kusanagi through a 500 µl pipette tip three to five times. Cells were et al. 2000, Korchynskyi & ten Dijke 2002, Mazerbourg then diluted in 9 ml 0·9% saline, and counted using a et al. 2004, Monteiro et al. 2004). Granulosa cells from Coulter counter (model Zm; Coulter Electronics, Hialeah, small follicles were cultured as described in experiment 1 FL, USA) as previously described (Langhout et al. 1991, with the following treatments applied for 24 h in medium Spicer & Chamberlain 1998, Spicer et al. 2002). (containing 1% FCS, 30 ng/ml IGF-I, and FSH) after a Concentrations of progesterone and estradiol in culture 4-h transfection with the hormone-specific responsive medium were determined by RIA as previously described promoters CAGA or BRE: control (no additions), GDF-9 (Langhout et al. 1991, Spicer et al. 2002). The intra- and (300 ng/ml), or BMP-4 (200 ng/ml). The doses of hor- interassay coefficients of variation were 12% and 17% www.endocrinology-journals.org Journal of Endocrinology (2006) 189, 329–339

Downloaded from Bioscientifica.com at 09/27/2021 08:08:57AM via free access 332 L J SPICER and others · Effects of GDF-9 in bovine granulosa cells

for the progesterone RIA, and 7% and 14% for the estradiol RIA.

Statistical analysis Each experiment contained three replicates per treatment and each experiment was replicated three to four times with different pools of granulosa cells. Each pool of small-follicle granulosa cells was obtained from multiple follicles collected from seven to ten cattle, and each pool of large-follicle granulosa cells was obtained from four to seven follicles collected from three to six cattle. Data are presented as the least squares means (S.E.) of measure- ments from nine to twelve culture wells. Main effects (i.e. hormone, dose, experimental replicate) and interactions were assessed using the general linear model procedure of SAS (1999). Steroid production was expressed as ng or pg/105 cells per 24 h, and cell numbers at the termination of the experiment were used for this calculation. Specific differences in cell numbers, steroid production, and pro- moter (luciferase) activity among treatments were deter- mined using Fisher’s protected least significant difference procedure (Ott 1977).

Results

Experiment 1: GDF-9 effects on small-follicle granulosa cells Cell numbers GDF-9 caused a dose-dependent increase (P<0·05) in basal (60% increase) and IGF-I-induced (28% increase) cell numbers (Fig. 1A).

Progesterone production IGF-I increased (P<0·001) progesterone production (to 2-fold of controls) by granu- losa cells, and GDF-9 caused a dose-dependent inhibition Figure 1 Stimulatory effect of 2-day treatment of various doses of (P<0·05) of this increase with 600 ng/ml completely GDF-9 in the presence of FSH (30 ng/ml) with or without IGF-I blocking the IGF-I-induced increase (Fig. 2A). At (30 ng/ml) on numbers of granulosa cells from small (1–5 mm) (A) ff and large (8–22 mm) (B) bovine follicles. Within a panel, means 150 ng/ml, GDF-9 had no significant e ect on progester- without a common letter differ (P<0·05; n=3 experiments). one production (Fig. 2A). In the absence of IGF-I, only 600 ng/ml decreased (by 28%; P<0·05) progesterone production in FSH-treated cultures (Fig. 2A). IGF-I-induced (maximum 2·3-fold increase) cell numbers (Fig. 1B). Estradiol production IGF-I increased (P<0·01) estra- diol production (to 20·7-fold of FSH-treated controls) by Progesterone production IGF-I increased (P<0·001) granulosa cells, and GDF-9 caused a dose-dependent progesterone production (to 2-fold of FSH-treated controls) decrease (P<0·05) in this IGF-I-induced estradiol produc- by granulosa cells, and GDF-9 caused a dose-dependent tion, with the highest dose tested (600 ng/ml) inhibiting inhibition (P<0·05) of this increase with 600 ng/ml com- estradiol production by 71% (Fig. 2B). At 300 ng/ml, pletely blocking the IGF-I-induced increase (Fig. 3A). GDF-9 inhibited (P<0·05) estradiol production by 27%. In contrast to small-follicle granulosa cells, 150 ng/ml In the absence of IGF-I, none of the doses of GDF-9 GDF-9 significantly decreased progesterone production affected (P>0·10) estradiol production in FSH-treated (Fig. 3A). In the absence of IGF-I but in the presence cultures (Fig. 2B). of FSH, only 600 ng/ml decreased (by 38%; P<0·05) progesterone production (Fig. 3A). Experiment 2: GDF-9 effects on large-follicle granulosa cells Cell numbers GDF-9 caused a dose-dependent increase Estradiol production IGF-I increased (P<0·01) estra- (P<0·01) in basal (maximum 2·1-fold increase) and diol production (to 7·7-fold of FSH-treated controls) by

Journal of Endocrinology (2006) 189, 329–339 www.endocrinology-journals.org

Downloaded from Bioscientifica.com at 09/27/2021 08:08:57AM via free access Effects of GDF-9 in bovine granulosa cells · L J SPICER and others 333

Figure 2 Inhibitory effect of 2-day treatment of various doses of Figure 3 Inhibitory effect of 2-day treatment of various doses of GDF-9 in the presence of FSH (30 ng/ml) with or without IGF-I GDF-9 in the presence of FSH (30 ng/ml) with or without IGF-I (30 ng/ml) on progesterone (A) and estradiol (B) production by (30 ng/ml) on progesterone (A) and estradiol (B) production by granulosa cells from small (1–5 mm) bovine follicles. Within a granulosa cells from large (8–22 mm) bovine follicles. Within a panel, means without a common letter differ (P<0·05; n=3 panel, means without a common letter differ (P<0·05; n=3 experiments). experiments). granulosa cells, and GDF-9 caused a dose-dependent 5·9-fold respectively (Fig. 4). In the presence of 1% and inhibition (P<0·05) of this increase with 300 and 2% FCS, GDF-9 further increased (P<0·05) cell numbers 600 ng/ml reducing the IGF-I-induced increase in estra- by 31% and 27% respectively (Fig. 4). diol production by 20% and 51% respectively (Fig. 3B). In the absence of IGF-I but in the presence of FSH, none Experiment 4: Effects of BMP-4 on small- and large-follicle of the doses of GDF-9 affected (P>0·10) estradiol granulosa cells production (Fig. 3B). Cell numbers BMP-4 had no effect (P>0·10) on ff numbers of granulosa cells collected from small (control= Experiment 3: E ect of GDF-9 on FCS-induced large-follicle 1·37 and BMP-4=1·540·04105 cells/well) or large granulosa cell growth (control=1·08 and BMP-4=1·220·06105 cells/well) Cell numbers GDF-9 amplified (P<0·05) basal and follicles. FCS-induced cell numbers (Fig. 4). Alone, GDF-9 increased (P<0·05) cells numbers by 2·1-fold whereas 1% Progesterone production BMP-4 inhibited (P<0·05) and 2% FCS increased (P<0·05) cell numbers by 4·7- and progesterone production induced by IGF-I and FSH by www.endocrinology-journals.org Journal of Endocrinology (2006) 189, 329–339

Downloaded from Bioscientifica.com at 09/27/2021 08:08:57AM via free access 334 L J SPICER and others · Effects of GDF-9 in bovine granulosa cells

Figure 4 Stimulatory effect of 2-day treatment of GDF-9 (300 ng/ml) on FCS-induced granulosa cell proliferation. Granulosa cells from large (8–22 mm) bovine follicles were cultured for 2 days in the presence of 0, 1, or 2% FCS. Means without a common letter differ (P<0·05; n=3 experiments).

39% and 27% in cultures of small- and large-follicle granulosa cells respectively (Fig. 5A).

Estradiol production BMP-4 inhibited (P<0·05) estra- diol production induced by IGF-I and FSH by 40% and 42% in cultures of granulosa cells from small and large follicles respectively (Fig. 5B).

Experiment 5: GDF-9 and BMP-4 effects on CAGA and BRE promoter activity in small-follicle granulosa cells Figure 5 Inhibitory effect of 2-day treatment of BMP-4 (30 ng/ml) Granulosa cells from small follicles were cultured for 48 h in the presence of FSH (30 ng/ml) and IGF-I (30 ng/ml) on in 10% FCS, followed by transfection with either the progesterone (A) and estradiol (B) production by granulosa cells from large (8–22 mm) and small (1–5 mm) bovine follicles. Means CAGA or BRE promoter and hormonal treatments re- without a common letter differ (P<0·05; n=4 experiments). vealed that treatment with GDF-9 increased (P<0·01) CAGA promoter activity 1·8-fold of controls, whereas BMP-4 was without effect (P>0·10; Fig. 6A). In contrast, Conditioned medium from 293T cells had no significant BMP-4 increased (P<0·01) BRE promoter activity by effect on progesterone production (Table 1). 3·9-fold of controls, whereas GDF-9 was without effect (P>0·10; Fig. 6B). Estradiol production FSH increased (P<0·05) estradiol production (to 4·7-fold of IGF-I-treated controls) by ff granulosa cells, and rhGDF-9 significantly reduced this Experiment 6: E ects of rhGDF-9 and conditioned medium FSH-induced increase by 40% (Table 1). Conditioned from 293T cells on small-follicle granulosa cells medium from 293T cells had no significant effect on Cell numbers rhGDF-9 but not FSH or conditioned estradiol production (Table 1). medium from 293T cells increased (P<0·05) the numbers of granulosa cells collected from small follicles (Table 1). Discussion Progesterone production FSH increased (P<0·05) progesterone production by 36% in the presence of The results of the present study on bovine granulosa cells IGF-I whereas rhGDF-9 inhibited (P<0·05) this FSH- revealed that: (1) GDF-9 stimulates the proliferation of induced increase in progesterone production (Table 1). granulosa cells from both large and small follicles in the

Journal of Endocrinology (2006) 189, 329–339 www.endocrinology-journals.org

Downloaded from Bioscientifica.com at 09/27/2021 08:08:57AM via free access Effects of GDF-9 in bovine granulosa cells · L J SPICER and others 335

presence and absence of IGF-I and FCS, whereas BMP-4 was without effect; (2) GDF-9 and BMP-4 inhibit FSH- and IGF-I-induced estradiol and progesterone production by granulosa cells from both large and small follicles; (3) GDF-9 has minimal effect on granulosa cell progesterone and estradiol production induced by FSH alone; (4) GDF-9 (but not BMP-4) activates the CAGA pro- moter; and (5) BMP-4 (but not GDF-9) activates the BRE promoter. In the present study, stimulatory effects of GDF-9 were observed on basal and IGF-I-induced granulosa cell pro- liferation whether cells were obtained from large or small follicles. These observations agree with previous reports in which treatment with GDF-9 (10–2000 ng/ml) increased numbers of rat granulosa cells (by over 2-fold) as well as cell proliferation (by 2- to 9-fold) as measured by [3H]thy- midine incorporation (Vitt et al. 2000a, 2002, McNatty et al. 2005a). In contrast, a recent report indicates that treatment with either 1 or 2 µg/ml recombinant ovine GDF-9 had no significant effect on [3H]thymidine incor- poration into ovine or bovine granulosa cells (McNatty et al. 2005b). In further agreement with the present study, the proliferative responses of rat granulosa cells to GDF-9 observed by Vitt et al. (2000a) was qualitatively similar whether cells were obtained from small antral follicles or large preovulatory follicles, although the maximally effec- tive dose of GDF-9 was 5-fold lower in rat granulosa cells obtained from large preovulatory follicles. The latter suggests that rat granulosa cells of mature differentiated follicles may be more sensitive to GDF-9 than those of immature undifferentiated follicles. Data in the present study support this assumption because large-follicle granu- losa cell proliferation and progesterone production (see next paragraph) were significantly affected by the lowest dose of GDF-9 tested, whereas small-follicle granulosa Figure 6 Stimulatory effect of 24-h treatment of GDF-9 (but not cells were not. Interestingly, BMP-4 at a dose effective in BMP-4) (A) on CAGA promoter activity and BMP-4 (but not ff GDF-9) (B) on BRE promoter activity in granulosa cells from small inhibiting steroidogenesis had no e ect on IGF-I-induced (1–5 mm) bovine follicles. Means without a common letter differ cell numbers in the present study. Shepherd & Nachtigal (P<0·05).
-Galac,
-galactosidase. (2003) reported that neither 2- nor 4-day BMP-4 treatment affected FCS-induced proliferation of four types

Table 1 Effects of recombinant human GDF-9 (rhGDF-9) and conditioned medium (CM: from non-transfected human embryonic kidney 293T cells) on granulosa cell numbers, and on progesterone and estradiol production

No. of Cell numbers Progesterone Estradiol samples (105/well) (ng/105 cells/24 h) (pg/105 cells/24 h) Treatment* Control 9 0·61a 42·0a 46a +FSH 9 0·77b 57·1b 216c +FSH +CM 9 0·84b 56·2b 248c +FSH +rhGDF-9 9 1·06c 37·9a 130b S.E.M. 0·05 4·3 17

Granulosa cells from small (1 to 5 mm) bovine follicles were cultured as described in Materials and Methods, and treated for 48 h with FSH (30 ng/mL), CM (2 l/well), and/or 600 ng/mL of rhGDF-9 . *All treatments were applied concomitantly with 30 ng/mL of IGF-I. abcWithin a column, means without a common letter differ (P<0·05). www.endocrinology-journals.org Journal of Endocrinology (2006) 189, 329–339

Downloaded from Bioscientifica.com at 09/27/2021 08:08:57AM via free access 336 L J SPICER and others · Effects of GDF-9 in bovine granulosa cells

of human ovarian carcinoma cells. Also, others have granulosa cells of the present study supports this reported that BMP-4 (Fabre et al. 2003) and BMP-2 latter suggestion. (Souza et al. 2002) have no effect on ovine granulosa cell Reported for the first time using bovine granulosa cells, proliferation while significantly affecting steroidogenesis GDF-9 inhibited estradiol secretion by granulosa cells in vitro. Thus, the present study supports the notion that obtained from large and small follicles of a single- two TGF-
superfamily members that bind to the same ovulating species (Figs 2B and 3B). Previously, treatment of BMPRII receptor (Kawabata et al. 1998, Lux et al. 1999, rat granulosa cells with GDF-9 (30–150 ng/ml) inhibited Newfeld et al. 1999) but derived from two different (by up to 58%) FSH-induced estradiol production with- (i.e. oocyte vs theca) cell types have diverse biological out affecting forskolin-induced estradiol production (Vitt responses within the granulosa layer, the latter of which et al. 2000a). Thus, granulosa cell aromatase activity from are mediated by the various type I receptors (ALKs) and multiple- and single-ovulating species appear to respond intracellular Smad response proteins (Massague 1998, similarly to GDF-9. Also consistent with the present Mazerbourg & Hsueh 2003, Mazerbourg et al. 2004). study, Vitt et al. (2000a) found that the inhibitory effect of Whether GDF-9 and BMP-4 have stimulatory effects on GDF-9 on estradiol production was similar whether cells preantral follicular growth in cattle as they do in rodents were obtained from small antral or large preovulatory rat (Vitt et al. 2000b, Nilsson & Skinner 2002, 2003, Latham follicles. Collectively, these studies indicate that GDF-9 is et al. 2004, Wang & Roy 2004) and humans (Hreinsson an effective suppressor of aromatase activity regardless of et al. 2002) awaits further elucidation. Also, whether the size (or differentiation state) of the follicle in which the GDF-9 plays a role in the regulation of ovulation rate in granulosa cells reside. cattle, as recently demonstrated for sheep (Hanrahan et al. Similar to GDF-9, BMP-4 inhibited both progesterone 2004, Juengel et al. 2004), will require additional research. and estradiol production by bovine granulosa cells from Previous studies with rat granulosa cells have reported small and large follicles. Although originally identified for that 48-h (Vitt et al. 2000a, 2002) and 6-day (McNatty their ability to induce bone and cartilage formation (Wan et al. 2005a) treatment with>30 ng/ml GDF-9 inhibits & Cao 2005), BMPs have since been shown to play progesterone production induced by FSH. Similarly, in important roles in cellular differentiation and formation the present study, GDF-9 decreased bovine granulosa cell in many other tissues and organs including the ovary progesterone production in the presence of FSH alone (Massague 1998, Knight & Glister 2003, Mazerbourg & (highest dose of GDF-9 only) or a combination of FSH Hsueh 2003). Inhibitory effects of BMP-4 on progester- and IGF-I (all doses of GDF-9). McNatty et al. (2005b) one production by granulosa cells of sheep (Mulsant et al. recently reported that 6-day treatment with 2 µg/ml 2001, Fabre et al. 2003, Pierre et al. 2004), cattle (Glister GDF-9 inhibited progesterone production by ovine and et al. 2004), and rats (Shimasaki et al. 1999, Sudo et al. bovine granulosa cells cultured in the presence of FSH, 2004) have been documented. The present study further IGF-I, and insulin. Yamamoto et al. (2002) also observed evaluated BMP-4 effects on aromatase activity. In cul- that GDF-9 inhibits progesterone production induced by tured rat granulosa cells, BMP-4 stimulates FSH-induced 8-bromo-cAMP in cultured human granulosa cells. In the estradiol production (Shimasaki et al. 1999, Sudo et al. present study, under basal conditions (the presence of 2004) but its effect in the presence of IGF-I was not FSH), GDF-9 (only 600 ng/ml) had a weak inhibitory studied. One other report indicated that a 6-day treatment effect on progesterone production. In previous studies of bovine granulosa cells with BMP-4 increases estradiol with cultured mouse granulosa cells, 50–100 ng/ml production and cell proliferation (Glister et al. 2004) but GDF-9 treatment for 16–24 h increased basal progester- because estradiol production was not corrected for one production but had no effect on FSH-induced increased cell numbers, clear interpretation of these data is progesterone production (Elvin et al. 1999, 2000). not possible. In the present study, BMP-4 suppressed FSH Discrepancies among studies could be due to species plus IGF-I-induced aromatase activity as well as progester- differences and/or differences in culture conditions (e.g. one production in bovine granulosa cells regardless of duration of treatment or presence or absence of FCS). whether cells originated from small or large follicles. More Consistent with previous reports (Spicer & Chamberlain extensive studies are needed to determine if responsiveness 1998, Spicer et al. 2002), IGF-I stimulated progesterone to BMP-4 changes during follicular development and and estradiol production by bovine granulosa cells in the if locally produced BMPs change during follicular presence of FSH (Figs 2 and 3). It should be emphasized development in cattle. that granulosa cells in the present and previous studies, Using bovine granulosa cells, we have shown that because of serum exposure (Orly et al. 1980, Luck et al. GDF-9 (but not BMP-4) activated the Smad3-induced 1990), may have partially luteinized and therefore be CAGA promoter, whereas BMP-4 (but not GDF-9) exhibiting some luteal cell activity. The less than 5-fold activated the Smad1/5/8-induced BRE promoter. Similar increase in estradiol secretion induced by a high dose (i.e. results has been obtained in rat and human granulosa 30 ng/ml) of FSH combined with the very high ratio cells (Kaivo-Oja et al. 2003, 2005, Roh et al. 2003, (i.e.>150) of progesterone:estradiol secreted by the Mazerbourg et al. 2004, Sudo et al. 2004). Previous studies

Journal of Endocrinology (2006) 189, 329–339 www.endocrinology-journals.org

Downloaded from Bioscientifica.com at 09/27/2021 08:08:57AM via free access Effects of GDF-9 in bovine granulosa cells · L J SPICER and others 337 in rat and human granulosa cells have suggested that Carabatsos MJ, Elvin J, Matzuk MM & Albertini DF 1998 GDF-9 induces the phosphorylation of Smad2/3 after Characterization of oocyte and follicle development in growth differentiation factor-9-deficient mice. Developmental Biology interaction with the receptor type II, BMPRII, and the 204 373–384. receptor type I, ALK5 (Vitt et al. 2002, Mazerbourg et al. Dennler S, Itoh S, Vivien D, ten Dijke P, HuetS&Gauthier JM 2004, Kaivo-Oja et al. 2005). Stimulation of the CAGA 1998 Direct binding of Smad3 and Smad4 to critical TGF promoter is mediated by TGF-
, activin, or GDF-9 after
-inducible elements in the promoter of human plasminogen phosphorylation of the downstream Smad3 proteins, activator inhibitor-type 1 gene. EMBO Journal 17 3091–3100. whereas BMP-2 and BMP-7 activate Smad1, 5, and 8 Dong J, Albertini DF, Nishimori K, Kumar TR, LuN&Matzuk MM 1996 Growth differentiation factor-9 is required during early (Massague 1998) and subsequently the BRE and GCCG ovarian folliculogenesis. Nature 383 531–535. promoters (Kusanagi et al. 2000, Korchynskyi & ten Dijke Elvin JA, Clark AT, Wang P, Wolfman NM & Matzuk MM 1999 2002). However, the distinct biological effects of the Paracrine actions of growth differentiation factor-9 in the TGF-
superfamily members may be exerted by the mammalian ovary. Molecular Endocrinology 13 1035–1048. transcriptional regulation of target genes in a cell-type Elvin JA, YanC&Matzuk MM 2000 Growth differentiation factor-9 specific manner that may involve ALK-specific and stimulates progesterone synthesis in granulosa cells via a ff prostaglandin E2/EP2 receptor pathway. PNAS 97 10288–10293. opposing e ects that may include the inhibitory Smads Erickson GF & Shimasaki S 2003 The spatiotemporal expression (Smad6 and Smad7) (ten Dijke & Hill 2004) and may pattern of the bone morphogenetic protein family in rat ovary cell explain how factors such as GDF-9 have both stimulatory types during the estrous cycle. Reproductive Biology and Endocrinology and inhibitory effects on granulosa cell functions. Further 1 9–29. studies will be required to elucidate the elements involved Fabre S, Pierre A, Pisselet C, Mulsant P, Lecerf F, Pohl J, Monget P in the inhibitory (steroidogenesis) versus stimulatory (cell & Monniaux D 2003 The Booroola mutation in sheep is associated with an alteration of the bone morphogenetic protein receptor-IB proliferation) action of GDF-9. functionality. Journal of Endocrinology 177 435–444. Fatehi AN, van den Hurk R, Colenbrander B, Daemen AJ, van Tol HT, Monteiro RM, Roelen BA & Bevers MM 2005 Expression of bone morphogenetic protein2 (BMP2), BMP4 and Acknowledgements BMP receptors in the bovine ovary but absence of effects of BMP2 and BMP4 during IVM on bovine oocyte nuclear maturation and The authors thank M Aleman and C Klein for technical subsequent embryo development. Theriogenology 63 872–889. assistance, Creekstone Farms (Arkansas City, KS, USA) Glister C, Kemp CF & Knight PG 2004 Bone morphogenetic protein for their generous donations of bovine ovaries, Dr C H (BMP) ligands and receptors in bovine ovarian follicle cells: actions ff Heldin (Ludwig Institute for Cancer Research, Uppsala, of BMP-4, -6 and -7 on granulosa cells and di erential modulation of Smad-1 phosphorylation by follistatin. Reproduction 127 239–254. Sweden) for the CAGA promoter–luciferase construct, Dr Hanrahan JP, Gregan SM, Mulsant P, Mullen M, Davis GH, O Korchynskyi (University of North Carolina, Chapel PowellR&Galloway SM 2004 Mutations in the genes for Hill, NC, USA) for the BRE promoter–luciferase con- oocyte-derived growth factors GDF9 and BMP15 are associated struct, and Dr R Matts (Oklahoma State University, with both increased ovulation rate and sterility in Cambridge and Stillwater, OK, USA) for use of the luminometer. Belclare sheep (Ovis aries). Biology of Reproduction 70 900–909. Hayashi M, McGee EA, Min G, Klein C, Rose UM, van Duin M & Hsueh AJ 1999 Recombinant growth differentiation factor-9 (GDF-9) enhances growth and differentiation of cultured early Funding ovarian follicles. Endocrinology 140 1236–1244. Hreinsson JG, Scott JE, Rasmussen C, Swahn ML, Hsueh AJ & Hovatta O 2002 Growth differentiation factor-9 promotes the This research was supported in part under project H-2510 growth, development, and survival of human ovarian follicles in of the Oklahoma Agricultural Experiment Station, and by organ culture. Journal of Clinical Endocrinology and Metabolism the National Research Initiative Competitive Grant no. 87 316–321. 2005–35203–15334 from the USDA Cooperative State Hunter MG, Robinson RS, Mann GE & Webb R 2004 Endocrine Research, Education, and Extension Service. This paper and paracrine control of follicular development and ovulation rate was approved for publication by the Director, Oklahoma in farm species. Animal Reproduction Science 82–83 461–477. Jaatinen R, Laitinen MP, Vuojolainen K, Aaltonen J, Louhio H, Agricultural Experimental Station. The authors declare Heikinheimo K, LehtonenE&RitvosO1999Localization of that there is no conflict of interest that would prejudice growth differentiation factor-9 (GDF-9) mRNA and protein in rat the impartiality of this scientific work. ovaries and cDNA cloning of rat GDF-9 and its novel homolog GDF-9B. Molecular and Cellular Endocrinology 156 189–193. Juengel JL & McNatty KP 2005 The role of proteins of the transforming growth factor-
superfamily in the intraovarian References regulation of follicular development. Human Reproduction Update 11 143–160. Bodensteiner KJ, McNatty KP, Clay CM, Moeller CL & Sawyer HR Juengel JL, Hudson NL, Heath DA, Smith P, Reader KL, 2000 Expression of growth and differentiation factor-9 in the Lawrence SB, O’Connell AR, Laitinen MP, Cranfield M, Groome NP ovaries of fetal sheep homozygous or heterozygous for the et al. 2002 Growth differentiation factor 9 and bone morphogenetic inverdale prolificacy gene (FecX(I)). Biology of Reproduction protein 15 are essential for ovarian follicular development in sheep. 62 1479–1485. Biology of Reproduction 67 1777–1789. www.endocrinology-journals.org Journal of Endocrinology (2006) 189, 329–339

Downloaded from Bioscientifica.com at 09/27/2021 08:08:57AM via free access 338 L J SPICER and others · Effects of GDF-9 in bovine granulosa cells

Juengel JL, Hudson NL, WhitingL&McNatty KP 2004 Effects of McGee EA & Hsueh AJ 2000 Initial and cyclic recruitment of ovarian immunization against bone morphogenetic protein 15 and growth follicles. Endocrine Reviews 21 200–214. differentiation factor 9 on ovulation rate, fertilization, and McGrath SA, Esquela AF & Lee SJ 1995 Oocyte-specific expression pregnancy in ewes. Biology of Reproduction 70 557–561. of growth/differentiation factor-9. Molecular Endocrinology Kaivo-Oja N, Bondestam J, Kamarainen M, Koskimies J, Vitt U, 9 131–136. Cranfield M, Vuojolainen K, Kallio JP, Olkkonen VM, Hayashi M McNatty KP, Juengel JL, Reader KL, Lun S, Myllymaa S, et al. 2003 Growth differentiation factor-9 induces Smad2 Lawrence SB, Western A, Meerasahib MF, Mottershead DG, activation and inhibin B production in cultured human Groome NP et al. 2005a Bone morphogenetic protein 15 and granulosa-luteal cells. Journal of Clinical Endocrinology and Metabolism growth differentiation factor 9 co-operate to regulate granulosa cell 88 755–762. function. Reproduction 129 473–480. Kaivo-Oja N, Mottershead DG, Mazerbourg S, Myllymaa S, Duprat S, McNatty KP, Juengel JL, Reader KL, Lun S, Myllymaa S, Gilchrist RB, Groome NP, Hsueh AJ & Ritvos O 2005 Adenoviral Lawrence SB, Western A, Meerasahib MF, Mottershead DG, gene transfer allows Smad-responsive gene promoter analyses and Groome NP et al. 2005b Bone morphogenetic protein 15 and delineation of type I receptor usage of transforming growth growth differentiation factor 9 co-operate to regulate granulosa cell factor-
family ligands in cultured human granulosa luteal cells. function in ruminants. Reproduction 129 481–487. Journal of Clinical Endocrinology and Metabolism 90 271–278. Monteiro RM, de Sousa Lopes SM, Korchynskyi O, ten Dijke P & Kawabata M, ImamuraT&Miyazono K 1998 Signal transduction by Mummery CL 2004 Spatio-temporal activation of Smad1 and bone morphogenetic proteins. Cytokine and Growth Factor Reviews Smad5 in vivo: monitoring transcriptional activity of Smad proteins. 9 49–61. Journal of Cell Science 117 4653–4663. Knight PG & Glister C 2003 Local roles of TGF-
superfamily Mulsant P, Lecerf F, Fabre S, Schibler L, Monget P, Lanneluc I, members in the control of ovarian follicle development. Animal Pisselet C, Riquet J, Monniaux D, Callebaut I et al. 2001 Mutation Reproduction Science 78 165–183. in bone morphogenetic protein receptor-IB is associated with Korchynskyi O & ten Dijke P 2002 Identification and functional increased ovulation rate in Booroola Merino ewes. PNAS characterization of distinct critically important bone morphogenetic 98 5104–5109. protein-specific response elements in the Id1 promoter. Journal of Newfeld SJ, Wisotzkey RG & Kumar S 1999 Molecular evolution of Biological Chemistry 277 4883–4891. a developmental pathway: phylogenetic analyses of transforming Kusanagi K, Inoue H, Ishidou Y, Mishima HK, Kawabata M & growth factor-
family ligands, receptors and Smad signal Miyazono K 2000 Characterization of a bone morphogenetic transducers. Genetics 152 783–795. protein-responsive Smad-binding element. Molecular Biology of the Nilsson EE & Skinner MK 2002 Growth and differentiation factor-9 Cell 11 555–565. stimulates progression of early primary but not primordial rat Langhout DJ, Spicer LJ & Geisert RD 1991 Development of a culture ovarian follicle development. Biology of Reproduction 67 1018–1024. system for bovine granulosa cells: effects of growth hormone, Nilsson EE & Skinner MK 2003 Bone morphogenetic protein-4 acts estradiol, and gonadotropins on cell proliferation, steroidogenesis, as an ovarian follicle survival factor and promotes primordial follicle and protein synthesis. Journal of Animal Science 69 3321–3334. development. Biology of Reproduction 69 1265–1272. Latham KE, Wigglesworth K, McMenamin M & Eppig JJ 2004 OrlyJ,SatoG&Erickson GF 1980 Serum suppresses the expression Stage-dependent effects of oocytes and growth differentiation factor of hormonally induced functions in cultured granulosa cells. Cell 9 on mouse granulosa cell development: advance programming and 20 817–827. subsequent control of the transition from preantral secondary follicles to early antral tertiary follicles. Biology of Reproduction Ott L 1977 Multiple comparisons. In An Introduction to Statistical 70 1253–1262. Methods and Data Analysis, pp 384–388. North Scituate, MA, USA: Lonergan P, Gutierrez-Adan A, Rizos D, Pintado B, de la Fuente J & Duxbury Press. Boland MP 2003 Relative messenger RNA abundance in bovine Pennetier S, Uzbekova S, Perreau C, Papillier P, Mermillod P & oocytes collected in vitro or in vivo before and 20 hr after the Dalbies-Tran R 2004 Spatio-temporal expression of the germ cell preovulatory luteinizing hormone surge. Molecular Reproduction and marker genes MATER, ZAR1, GDF9, BMP15, and VASA in Development 66 297–305. adult bovine tissues, oocytes, and preimplantation embryos. Biology Luck MR, Rodgers RJ & Findlay JK 1990 Secretion and gene of Reproduction 71 1359–1366. expression of inhibin, oxytocin and steroid hormones during the Pierre A, Pisselet C, Dupont J, Mandon-Pepin B, Monniaux D, in vitro differentiation of bovine granulosa cells. Reproduction, Monget P & Fabre S 2004 Molecular basis of bone morphogenetic Fertility and Development 2 11–25. protein-4 inhibitory action on progesterone secretion by ovine Lux A, AttisanoL&Marchuk DA 1999 Assignment of transforming granulosa cells. Journal of Molecular Endocrinology 33 805–817. growth factor
1and
3 and a third new ligand to the type I Prochazka R, Nemcova L, NagyovaE&Kanka J 2004 Expression of receptor ALK-1. Journal of Biological Chemistry 274 9984–9992. growth differentiation factor 9 messenger RNA in porcine growing Marion GB, Gier HT & Choudary JB 1968 Micromorphology of and preovulatory ovarian follicles. Biology of Reproduction the bovine ovarian follicular system. Journal of Animal Science 71 1290–1295. 27 451–465. Roh JS, Bondestam J, Mazerbourg S, Kaivo-Oja N, Groome N, Massague J 1998 TGF-
signal transduction. Annual Reviews of RitvosO&HsuehAJ2003Growthdifferentiation factor-9 Biochemistry 67 753–791. stimulates inhibin production and activates Smad2 in cultured rat MazerbourgS&HsuehAJ2003 Growth differentiation factor-9 granulosa cells. Endocrinology 144 172–178. signaling in the ovary. Molecular and Cellular Endocrinology SAS 1999 The SAS System for Windows, version 8. Cary, NC, USA: 202 31–36. SAS Institute Inc. Mazerbourg S, Bondy CA, Zhou J & Monget P 2003 The Sendai Y, Itoh T, Yamashita S & Hoshi H 2001 Molecular cloning of insulin-like growth factor system: a key determinant role in the a cDNA encoding a bovine growth differentiation factor-9 growth and selection of ovarian follicles? A comparative species (GDF-9) and expression of GDF-9 in bovine ovarian oocytes and study. Reproduction in Domestic Animals 38 247–258. in vitro-produced embryos. Cloning 3 3–10. Mazerbourg S, Klein C, Roh J, Kaivo-Oja N, Mottershead DG, Shepherd TG & Nachtigal MW 2003 Identification of a putative KorchynskyiO,RitvosO&HsuehAJ2004 Growth autocrine bone morphogenetic protein-signaling pathway in human differentiation factor-9 signaling is mediated by the type I receptor, ovarian surface epithelium and ovarian cancer cells. Endocrinology activin receptor-like kinase 5. Molecular Endocrinology 18 653–665. 144 3306–3314.

Journal of Endocrinology (2006) 189, 329–339 www.endocrinology-journals.org

Downloaded from Bioscientifica.com at 09/27/2021 08:08:57AM via free access Effects of GDF-9 in bovine granulosa cells · L J SPICER and others 339

Shimasaki S, Zachow RJ, Li D, Kim H, Iemura S, Ueno N, Sudo S, Avsian-Kretchmer O, Wang LS & Hsueh AJ 2004 Protein Sampath K, Chang RJ & Erickson GF 1999 A functional related to DAN and cerberus is a bone morphogenetic protein bone morphogenetic protein system in the ovary. PNAS antagonist that participates in ovarian paracrine regulation. Journal of 96 7282–7287. Biological Chemistry 279 23134–23141. Souza CJ, Campbell BK, McNeilly AS & Baird DT 2002 Effect of tenDijkeP&HillCS2004Newinsights into TGF-
-Smad bone morphogenetic protein 2 (BMP2) on oestradiol and inhibin A signalling. Trends in Biochemical Sciences 29 265–273. production by sheep granulosa cells, and localization of BMP Vitt UA, Hayashi M, KleinC&Hsueh AJ 2000a Growth receptors in the ovary by immunohistochemistry. Reproduction differentiation factor-9 stimulates proliferation but suppresses the 123 363–369. follicle-stimulating hormone-induced differentiation of cultured Spicer LJ 2004 Proteolytic degradation of insulin-like growth factor granulosa cells from small antral and preovulatory rat follicles. binding proteins by ovarian follicles: a control mechanism for Biology of Reproduction 62 370–377. selection of dominant follicles. Biology of Reproduction Vitt UA, McGee EA, Hayashi M & Hsueh AJ 2000b In vivo treatment 70 1223–1230. with GDF-9 stimulates primordial and primary follicle progression and theca cell marker CYP17 in ovaries of immature rats. Spicer LJ & Francisco CC 1997 The adipose obese gene product, Endocrinology 141 3814–3820. leptin: evidence of a direct inhibitory role in ovarian function. Vitt UA, Mazerbourg S, KleinC&Hsueh AJ 2002 Bone Endocrinology 138 3374–3379. morphogenetic protein receptor type II is a receptor for growth Spicer LJ & Chamberlain CS 1998 Influence of cortisol on insulin- differentiation factor-9. Biology of Reproduction 67 473–480. and insulin-like growth factor 1 (IGF-1)-induced steroid WanM&CaoX2005BMPsignaling in skeletal development. production and on IGF-1 receptors in cultured bovine granulosa Biochemical and Biophysical Research Communications 328 651–657. cells and thecal cells. Endocrine 9 153–161. WangJ&RoySK2004Growthdifferentiation factor-9 and stem cell Spicer LJ, Chamberlain CS & Morgan GL 2001 Proteolysis of factor promote primordial follicle formation in the hamster: insulin-like growth factor binding proteins during preovulatory modulation by follicle-stimulating hormone. Biology of Reproduction follicular development in cattle. Domestic Animal Endocrinology 70 577–585. 21 1–15. Yamamoto N, Christenson LK, McAllister JM & Strauss JF III 2002 Spicer LJ, Chamberlain CS & Maciel SM 2002 Influence of Growth differentiation factor-9 inhibits 35-adenosine gonadotropins on insulin- and insulin-like growth factor-I monophosphate-stimulated steroidogenesis in human granulosa (IGF-I)-induced steroid production by bovine granulosa cells. and theca cells. Journal of Clinical Endocrinology and Metabolism Domestic Animal Endocrinology 22 237–254. 87 2849–2856. Stewart RE, Spicer LJ, Hamilton TD, Keefer BE, Dawson LJ, Morgan GL & Echternkamp SE 1996 Levels of insulin-like growth Received in final form 4 February 2006 factor (IGF) binding proteins, luteinizing hormone and IGF-I receptors, and steroids in dominant follicles during the first Accepted 23 February 2006 follicular wave in cattle exhibiting regular estrous cycles. Made available online as an Accepted Preprint Endocrinology 137 2842–2850. 24 February 2006

www.endocrinology-journals.org Journal of Endocrinology (2006) 189, 329–339

Downloaded from Bioscientifica.com at 09/27/2021 08:08:57AM via free access