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Enhanced Response of Granulosa and Theca Cells from Sheep Carriers of the FecB Mutation in Vitro to Gonadotropins and Bone Morphogenic Protein-2, -4, and -6

Department of Obstetrics and Gynaecology (B.K.C., A.J.S.), Queen’s Medical Centre, University of Nottingham, Nottingham NG7 2UH, United Kingdom; Embrapa Pecuaria Sul (C.J.H.S.), Bage RS 96401-970, Brazil; School of Biosciences (R.W.), Sutton Bonington Campus, University of Nottingham LE12 5RD, United Kingdom; and Centre for Reproductive Biology (D.T.B.), University of Edinburgh, Edinburgh EH16 4SA, Scotland, United Kingdom

Address all correspondence and requests for reprints to: B. K. Campbell, School of Human Development, University of Nottingham, D Floor, East Block, Queen’s Medical Centre, Nottingham NG7 2UH, United Kingdom. E-mail: bruce.campbell{at}nottingham.ac.uk.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The FecB (Booroola) mutation, which leads to increased ovulation rates and multiple births in sheep, is now known to occur in the signaling domain of the bone morphogenic protein (BMP)-1B receptor. We examined the effect of the mutation on the responsiveness of granulosa (GC) and theca cells (TC) to BMPs and other local regulators using tissue from animals with (Fec^B/B) and without (Fec^+/+) the FecB mutation. Experiments examined the effect of BMP-2, -4, and -6 (0.005–50 ng/ml), and their interaction with IGF-I (0.1–10 ng/ml LR3 analog) and gonadotropins, on the proliferation and differentiation of GCs and TCs isolated from small (<2 mm) antral follicles and maintained in serum-free culture for up to 8 d. Dose-finding studies using ovaries from wild-type sheep obtained from the abbattoir showed no difference among the different BMPs in stimulating (P < 0.001) estradiol (E2) production by GCs cultured with FSH (10 ng/ml), but there was a clear interaction (P < 0.001) with IGF-I. BMPs had no effect on GC proliferation or the sensitivity of GCs to FSH. In contrast, higher doses of BMPs (5–50 ng/ml) inhibited LH-stimulated androstenedione production by TCs, whereas lower doses (0.005–0.05 ng/ml) stimulated TC proliferation (P < 0.01). Regardless of dose of IGF-I, at the end of culture (96–192 h) hormone production by GCs (E2, inhibin A) and TCs (androstenedione) was 4- to 5-fold greater (P < 0.001) by cells from Fec^B/B, compared with Fec^+/+ ewes exposed to the same dose of gonadotropin. In the presence of low concentrations of IGF-I (0.1 ng/ml), the maximum increase in the production of E2 and inhibin A by GCs from FF ewes in response to BMPs was observed at doses that were 3- to 10-fold lower (3–10 ng/ml) than ++ (30 ng/ml; P < 0.001). Low doses of BMPs stimulated proliferation of TCs from ++ (P < 0.01) but not FF ewes. Immunohistochemistry confirmed BMP-6 protein expression in the oocyte, granulosa, and thecal layers of antral follicles from both genotypes. These results confirm a major role for BMPs in controlling ovarian somatic cell function in sheep and provide evidence to support the hypothesis that the FecB mutation increases the BMP response of somatic cells when stimulated to differentiate by gonadotropins.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE FecB (Booroola) mutation in sheep results in dysregulation of the normal mechanisms of follicle selection in this species and has been the subject of intensive research for more than 30 yr (1, 2, 3). The recent discovery that this (4, 5, 6) and other multiovulatory phenotypes are due to mutations in components of the bone morphogenic protein (BMP) system (2, 7) suggests that this system may be one of the main ways in which follicle selection is regulated in monovulatory species. Whereas the exact mechanisms regulating the selection of ovulatory follicles from a much larger follicular cohort remain to be fully elucidated, it is thought that the basis of both recruitment and selection lies in the pattern of gonadotrophic stimulation received by the ovary and intrafollicular factors that modulate gonadotrophic actions (8, 9, 10, 11). Over the last decade, intensive research in this area has led to the identification of a number of key local regulators such as the IGF (2) and inhibin/activin (3) systems, that have been shown to modulate the sensitivity of follicular somatic cells to gonadotropins and are therefore considered to be central to the mechanism of follicle selection. Recent research has identified BMPs as other potentially important local factors that may interact with the other well-described regulators.

To date, most studies examining the role of the BMP system in ovarian physiology have been performed in polyovular rodents (12, 13, 14). The mRNAs encoding BMP-2, -3, -3b, -4, -6, -7, -15, growth differentiation factor (GDF)-9, and BMP receptor (BMPR)-IA, BMPR-IB, and BMPR-II have been identified in the ovary in various mammals (12). In rats, BMP-4 and -7 have been shown to colocalize to the theca interstitial cells and expression levels for both BMPs change during folliculogenesis (14). In the mouse, BMP-6 is expressed at high levels in immature and mature oocytes and at lower levels in the granulosa cells (15). The expression of BMP-15 (15, 16, 17, 18, 19) and GDF-9 (15, 17) is confined exclusively to the oocyte in most species studied. In human ovaries, BMP-3 mRNA is strongly detectable by Northern analysis, whereas BMP-2 mRNA level is low and BMP-3b undetectable (20). The rat (14), bovine (21), and sheep (22) ovary has also been shown to be a major site of BMPR-I and -II expression, supporting the hypothesis that BMPs are important to ovarian physiology. Cell culture studies in rats have shown that both BMP-4 and -7 caused an increase in FSH-induced estradiol production but a decrease in FSH-induced progesterone production (14). BMP-6 was also found to be potent in the attenuation of FSH-induced progesterone production; however, in contrast to BMP-4 and -7, BMP-6 did not alter FSH-induced estradiol production (23). BMP-15 is a potent stimulator of GC proliferation (18) and inhibitor of FSH receptor expression in rat granulosa cells (24). In contrast, GDF-9 promotes GC proliferation and inhibits FSH-induced steroidogenesis and LH receptor expression (25).

In sheep, naturally occurring genetic mutations in several components of the BMP system have been shown to induce increases in ovulation rate. In Inverdale (FecX^I) and Hanna (FecX^H) ewes, separate point mutations were identified in the BMP (BMP-15) gene on the X chromosome corresponding to sites in the mature peptide coding region of the BMP15 growth factor (26). A remarkable characteristic of these mutations is that those that are heterozygous for the FecX^I or FecX^H mutation have higher-than-normal ovulation rates and litter sizes, whereas the homozygotes are sterile (27). Similarly, in Cambridge and Belclare ewes, mutations in both BMP-15 and the closely related GDF-9 lead to marked increases in ovulation rate (28). In contrast, in Booroola (FecB), Garole (FecB), and Javanese (FecB) sheep, a point mutation has been identified in the highly conserved intracellular serine threonine kinase signaling domain of the BMPR-IB on chromosome 6 (4, 5, 6, 29). In FecB animals, a single A to G transition occurs at nucleotide position 830, thereby substituting an arginine for glutamine at position 249 of the protein. The consequences of this mutation are that heterozygote females have an ovulation rate around one or two times higher than the noncarriers, and homozygotes (FF) have ovulation rates around three to 10 times higher than noncarriers (1, 7), but the mechanisms underlying this effect are unknown.

The FecB mutation results in a dysregulation of the follicle selection mechanisms with the precocious development of a large number of small antral follicles, leading to the greatly increased ovulation rates and multiple births (30, 31, 32). Although the identity of the FecB mutation is now known, a major question remains in terms of whether the mutation acts at the level of the ovary or the pituitary. The BMPR-1B is expressed by the pituitary (5) in sheep, and a number of investigators have reported increased FSH in ewes with the FecB mutation (33, 34). Conversely, mRNA (5) and protein (4) expression for type I and II BMPRs has been demonstrated in sheep ovarian somatic and germ cells, and BMP-2 (22), BMP-4, and GDF-5 (6) have all been shown to modulate steroid production by sheep granulosa cells in culture. We recently examined this question using ovarian autotransplants with and without the FecB mutation treated with a potent GnRH antagonist for 3 wk to render the animals hypogonadotrophic and then stimulated with the same gonadotropin regimen designed to mimic the normal pattern of FSH and LH during the follicular phase (35). In this study, the difference in ovulation rate and the characteristic phenotype of smaller ovulatory follicles and corpus luteum was retained, providing strong evidence that the FecB mutation acts at the level of the ovary to modulate gonadotrophic responsiveness.

The current evidence therefore suggests that BMPs can augment FSH-stimulated granulosa cell differentiation in sheep, and this is a possible mechanism to explain the effect of the FecB mutation in inducing precocious maturation of ovarian follicles. However, much more work is needed to identify the physiological ligands of the ovarian BMPRs and determine their relative actions, potencies, and interactions with other local modulators in the control of ovarian somatic cell differentiation in monovulatory species. Furthermore, the effect of the FecB mutation on the responsiveness of granulosa and theca cells to stimulation by gonadotropins, BMPs, and other local regulators needs to be determined to understand how a single mutation can have such a profound effect on ovarian function. The aim of the current work was to examine these questions using serum-free culture systems for both sheep granulosa (36) and theca (37) cells, which allow induction of cellular differentiation in vitro in response to physiological doses of gonadotropins and local factors over time frames that parallel those observed for similar processes in vivo. The specific hypothesis being investigated was that the FecB mutation would result in an alteration in the sensitivity of ovarian granulosa and thecal cells to stimulation with gonadotropins (FSH/LH), BMPs, and other established differentiative (IGF-I) and proliferative (TGF) growth factors.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Experimental animals
Experiments were conducted in accordance with the Animal (Scientific Procedures) Act of 1986 (United Kingdom). For experiments examining the effect of the FecB mutation, experimental animals were Scottish Blackface Merino cross ewes obtained from an experimental flock that had been derived in such a way that the animals differed at the Booroola locus (being either Fec^+/+ or Fec^B/B) but were as similar as possible for the rest of the genome to provide experimental material with little risk of biased comparisons (38). The animals were run under field conditions and fed a maintenance diet consisting of concentrates and hay ad libitum. Ovarian tissue was collected during the midbreeding season on d 4–10 of the estrous cycle following synchronization using progestagen sponges (medroxyprogesterone-acetate; Dunlop, Dumfries, UK) and cloprostenol (Estrumate, Dunlop) as previously described (39). FecB animals were killed by anesthetic overdose and both ovaries recovered by midventral incision under sterile conditions. For dose-finding studies, ovaries were obtained from the local abbattoir, following stunning and exsanguination, as previously described (36). For expression studies, ovarian tissue was immediately fixed in 4% paraformaldehyde following excision, whereas for culture studies the ovaries were placed in dissection medium (see below) at 37 C.

Hormones and reagents
Human recombinant BMP-2, -4, and -6 for in vitro studies were obtained from Sigma-Aldrich (Poole, Dorset, UK). Mouse antihuman BMP-6 was obtained from Chemicon Europe (Hampshire, UK). Ovine (o) FSH and LH were obtained from the National Institutes of Health. All other reagents were obtained from Sigma-Aldrich unless otherwise stated.

Expression of BMP-6 protein in the sheep ovary
Ovaries from wild-type and FecB mutant animals were fixed overnight in freshly prepared 4% paraformaldehyde in PBS (pH 7.4), and after fixation, ovaries were cut longitudinally in two or four slices, care being taken to cut through the largest follicles on each ovary (40). The tissue was then dehydrated by passage through a graded series of alcohols and embedded in paraffin wax.

Immunohistochemistry
Ovarian 5-µm sections on SuperFrost Plus microscope slides (Menzel-Glaser, Braunschweig, Germany) were dewaxed in xylene for 2 x 3 min and rehydrated in decreasing concentrations of alcohol (90, 70, 30, and distilled water), followed by two washes of 5 min each in 0.01 M PBS (pH 7.4). Antigen retrieval was carried out by microwaving the sections in preboiled citrate buffer [0.01 M (pH 6.0)] in an 800-W microwave at 50% power for 15 min. The slides were then left to rest in the buffer for 20 min until cool. After two 5-min washes in PBS, the sections were incubated in 3% hydrogen peroxide in water for 10 min. After a further two 5-min washes in PBS, the sections were incubated in blocking buffer, consisting of 1.5% (vol/vol) normal horse serum (Vectastain kit; Vector Laboratories, Peterborough, UK) in PBS for 20 min and then blotted before incubation in a 1 µg/ml dilution of mouse anti-BMP-6 monoclonal antibody in PBS supplemented with 0.25% BSA (Sigma) overnight at 4 C. Negative controls were carried out by replacing the primary antibody with a 1:2352 dilution of mouse immunoglobulin (Sigma). Specificity was further confirmed by coincubating some slides with the primary antibody plus 2 µg/ml recombinant human (rh) BMP-6 overnight at 4 C. Following a further two 5-min washes in PBS, the sections were incubated in a 1:200 dilution of biotinylated horse antimouse immunoglobulin (Vectastain kit, Vector Laboratories) in blocking buffer for 20 min at room temperature. The sections were again washed twice in PBS for 10 min before incubation in the avidin-biotin peroxidase complex (Vectastain kit, Vector Laboratories) for 30 min at room temperature. After a final wash in PBS, bound antibodies were visualized as brown staining by a 5-min incubation in 3–3'diaminobenzidine (Vector Laboratories). The sections were counterstained with Mayer’s hematoxylin (NuStain, Nottingham, UK), dehydrated through increasing concentrations of alcohols and mounted using DPX mounting medium (NuStain) and coverslips. The slides were observed using a DMRB microscope (Leica, Wetzlar, Germany) for evidence of specific staining and assessed by two independent observers who classified staining in the theca, membrane granulose, and oocyte (if present) of primary/secondary (one to two layers of cuboidal granulosa cells), multilaminer preantral, small antral (<1 mm diameter), and antral follicles (1–6 mm diameter) on a scale of 1–6 with 1 being no staining, 2 being very light staining, 3 being light staining, 4 being medium staining, 5 being heavy staining, and 6 being very heavy staining. A minimum of 10 follicles per class were assessed in this way and the mean value from the two observers used for subsequent analysis.

Serum-free culture of granulosa and theca cells
The methodologies used for the collection and culture of sheep granulosa (36) and theca (37) cells have been previously described. Briefly, small (< 3.5 mm in diameter) ovarian follicles were dissected from ovaries collected from the abattoir in Medium 199 containing 20 mmol HEPES l^–1, 100 kIU penicillin l^–1, 0.1 µg streptomycin l^–1, and 1 mg amphotericin (Fungizone) l^–1 at 37 C with specific attention being given to the removal of all extraneous stromal tissue from the follicle wall. Small follicles were hemisected in Dulbecco’s PBS without calcium or magnesium and the follicle halves flushed repeatedly up and down the barrel of a 1-ml syringe. The thecal shells were allowed to settle, the granulosa cell rich supernatant removed, and the flushing procedure repeated. The antral fluid of large follicles was removed using a 1-ml syringe and 23G needle before the follicles were hemisected and granulosa cells removed by gentle scraping with an inoculation loop. The granulosa cell were washed twice in culture medium (McCoy’s 5a supplemented 100 kIU penicillin l^–1, 0.1 µg streptomycin l^–1, 3 mmol L-glutamine l^–1, 0.1% BSA (wt/vol), 2.5 mg transferrin l^–1, 4 µg selenium l^–1, 10^–7 mol androstenedione l^–1, and 10 ng/ml bovine insulin) before plating at a density of 75,000 viable cells/well into preprepared and equilibrated 96-well plates containing 200 µl culture medium.

The thecal shells were dispersed in an enzyme mix containing 5 g collagenase l^–1, 1 g hyaluronidase l^–1, 1 g protease l^–1, 2 g deoxyribonuclease l^–1, and 0.002% donor calf serum (vol/vol) in 20 ml PBS for 30–45 min at 37 C with gentle agitation. The reaction was stopped by the addition of 2 ml donor calf serum and the cells washed twice in culture medium (DMEM-F12 with 100 kIU penicillin l^–1, 0.1 µg streptomycin l^–1, 3 mmol L-glutamine l^–1, 0.1% BSA (w/vol), 2.5 mg transferrin l^–1, 4 µg selenium l^–1, 10 ng/ml bovine insulin, and 10 ng/ml LR3 IGF-I) before plating at a density of 75,000 viable cells/well (<1% contamination by granulosa cells) into preprepared and equilibrated 96-well plates containing 200 µl culture medium.

Cells were cultured in a humidified atmosphere with 5% carbon dioxide in air at 37 C. Granulosa and theca cells were cultured for a total of 8 and 6 d, respectively, with medium being changed at 48-h intervals. To minimize disturbance of the cells, only 175 µl media were gently removed and replaced at each change. The spent medium was stored at –20 C before assay. At the end of culture, the number of viable cells/well was estimated using neutral red uptake and the results expressed as nanograms of hormone produced per 10,000 viable cells per 48 h.

Experimental design
Effects of different BMPs and interaction with IGF-I and gonadotropins.
These experiments used tissue from wild-type animals obtained from the abbattoir to examine the dose-responsive effects of BMP-2, -4, and -6 on gonadotropin-induced granulosa and theca cell proliferation and differentiation and the interaction of these BMPs with the dose of FSH and IGF-I. Initial experiments tested the effect of BMP-6 (5 ng/ml) on the responsiveness and sensitivity of granulosa cells from small follicles to stimulation with FSH (0.001–10 ng/ml) and the interaction of BMP-6 with the differentiative factor IGF-I LR3 (1 ng/ml). Dose-response curves (0–50 ng/ml) for BMP-2, -4, and -6 were then established in granulosa and theca cells with a constant dose of gonadotropin previously shown to induce cellular differentiation (10 ng/ml oFSH-16 for granulosa and 0.1 ng/ml oLH-26 for theca) and a range of doses of IGF-I LR3 (0, 0.1, 1, and 10 ng/ml) previously shown to modulate somatic cell differentiation and proliferation (36, 37). Insulin concentrations were maintained at a dose of 10 ng/ml throughout. For these experiments, within cultures each dose combination was replicated in quadruplicate and each culture was repeated at least three times.

Effect of FecB mutation
These experiments examined the differential sensitivity of granulosa and theca cells from wild-type animals and ewes carrying the FecB mutation to FSH; LH; IGF-1 LR3; TGF; and BMP-2, -4, and -6. A common observation in FecB carriers is a change in the follicular hierarchy, and thus, for the gonadotropin dose-response studies, follicles were divided into three size classes: small (1–2.5 mm diameter), medium (2.5–4.5 mm), and large (>4.5 mm diameter), and where sufficient cells were available dose responses were established for FSH (0–10 ng/ml oFSH) and LH (0–10 ng/ml oLH-26) at constant doses of IGF-I LR3 (10 ng/ml) and insulin (10 ng/ml). IGF-I LR3 dose responses (0–10 ng/ml) used optimum doses of insulin (10 ng/ml) and FSH (10 ng/ml), whereas TGF dose responses used optimum doses of FSH (10 ng/ml), insulin (10 ng/ml), and IGF LR3 (10 ng/ml). For BMP dose-response studies, four doses (0, 1, 10, and 30 ng/ml) were used for each BMP with two doses of IGF-I LR3 (0.1 and 10 ng/ml), with a constant dose of gonadotropin (10 ng/ml oFSH and 0.1 ng/ml oLH-26) and insulin (10 ng/ml). Due to the rarity of these animals and the need to breed replacements, these cultures were performed as sufficient numbers become available from 1995 onward. In each year, six to eight ewes per line were killed to generate sufficient tissue for two replicate cultures established on successive days that compared the response of cells from the two lines with a particular factor. For these experiments, within cultures each dose combination was replicated at least in triplicate, and each culture was repeated at least twice with data being blocked by replicate culture for analysis.

Assays
Concentrations of estradiol (36), androstenedione (37), and inhibin A (41) in unextracted culture media were determined using previously described RIAs. For inhibin A measurements, culture media had to be diluted between 1:10 and 1:1000 in assay buffer. The sensitivity of the assays for estradiol, androstenedione, progesterone, and rh inhibin A were 50 pmol/liter, 175 pmol/liter, 380 pmol/liter, and 30 ng/liter, respectively. The intra- and intercoefficients of variation for all assays were less than 15%.

Statistical analysis
With the exception of the time-course data, all hormone production data were expressed as amount of hormone produced per 48 h per 10,000 cells (10 kcells). Because accurate cell number data were not available at 96 and 144 h of culture, the time-course data were expressed as a concentration. Following tests for normality and homogeneity, the significance of treatment effects was determined by ANOVA using replicate cultures as blocks, both within and across different years. Individual comparisons between treatments were made using Bonferroni’s test. For immunohistochemistry intensity scores, data were not normally distributed and was therefore analyzed by either Mann-Whitney U or Kruskal-Wallis H test, as was appropriate, using SPSS (Chicago, IL).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization studies
Granulosa cells.
Experiments designed to test the effect of BMP on the responsiveness of granulosa cells from small follicles to FSH, revealed an additive effect of BMP and IGF-I on FSH-stimulated estradiol production (Fig. 1). Thus, addition of BMP-6 (5 ng/ml) or IGF-I (1 ng/ml) to granulosa cells cultured in the presence of insulin (10 ng/ml) resulted in identical FSH dose response curves, whereas combined addition of both factors resulted in a 2-fold increase in the response of the cells to effective doses of FSH (Fig. 1). It is also notable from this experiment that BMP-6 had no effect on the sensitivity of the granulosa cells to FSH with only doses of 1 and 10 ng/ml, resulting in a significant induction of aromatase activity as measured by estradiol production. As previously described (36), FSH in the presence of insulin alone induced a 70% (P < 0.01) increase in cell number with an ED₅₀ of 0.01 ng/ml. BMP-6 alone had no effect on this dose response, whereas addition of IGF-I, with or without BMP-6, resulted in maximal cell numbers at all doses of FSH (data not shown).

View larger version (21K): [in this window] [in a new window]   FIG. 1. Mean ± SEM estradiol production by granulosa cells after 192 h culture with increasing doses of oFSH in the presence of 10 ng/ml insulin (full triangle); insulin and 5 ng/ml BMP-6 (open circle); insulin and 10 ng/ml IGF-I LR3 (closed circle); or insulin, BMP-6, and IGF-I LR3 combined (open squares). Column represents overall mean production in presence of all factors in the absence of FSH. Asterisks indicate significant difference from zero dose of FSH: *, P < 0.05; **, P < 0.01; and ***, P < 0.001. , Significant difference between BMP and IGF supplementation alone, compared with combined treatment at a given dose of FSH with , P < 0.01.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Mean ± SEM estradiol production by granulosa cells after 192 h culture with increasing doses of BMP in the presence of 10 ng/ml insulin and FSH (open square); insulin, FSH, and 0.1 ng/ml IGF-1 LR3 (full diamond); insulin, FSH, and 1 ng/ml IGF-I LR3 (open circle); and insulin, FSH, and 10 ng/ml IGF-I LR3 (closed triangle). Estradiol production data from cultures using BMP-2, -4, and -6 have been pooled due to lack of affect of type of BMP (ANOVA). A marked interaction between dose of BMP and IGF-I is clearly evident from the data (P < 0.05), with a flattening of the BMP dose-response curve with increasing dose of IGF-I. Asterisks indicate significant difference from zero dose of BMP within each dose of LR3 IGF-I: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

Theca cells.
In marked contrast to granulosa cells, all BMPs had a highly significant inhibitory effect on androstenedione production (Fig. 3A; P < 0.001) by theca cells. In this instance, however, there was a significant interaction between type and dose of BMP (P < 0.01), with BMP-6 being less potent than BMP-2 and -4 (Fig. 3A). There was no interaction between dose of IGF and type (P = 0.8) or dose of BMP (P = 0.7) for androstenedione production. In addition, low doses of BMPs stimulated a marked increase (P < 0.001) in theca cell number after 6 d of culture (Fig. 3B). There was no difference between types of BMP in the magnitude of this effect (P = 0.3). As previously shown (37), a dose of IGF-I had a highly significant effect on cell number (P < 0.001), but there was no interaction between dose of IGF and type (P = 0.8) or dose of BMP (P = 0.8). The combined effect of increased cell number and decreased production per cell resulted in an increase (P < 0.05) in total steroid production at the lowest doses of BMP-4 and -6 and a highly significant depression (P < 0.001) at the highest doses (Fig. 3C).

View larger version (29K): [in this window] [in a new window]   FIG. 3. BMP in thecal cells: effect of BMP-2 (open bars), BMP-4 (hatched bars), and BMP-6 (closed bars) on androstenedione production corrected for cell number (A), cell number (B), and total androstenedione production (C) by thecal cells after 144 h of culture. Stippled bar indicates production in the absence of any BMP and asterisks indicate significant difference from zero dose of BMP: *, P < 0.05, **, P < 0.01 and ***, P < 0.001. Values are mean ± SEM.

View larger version (147K): [in this window] [in a new window]   FIG. 4. Representative images showing BMP-6 protein expression in the sheep ovary. Strong expression was observed in the oocyte, cumulus and membrana granulosa cell layer at all stages of follicle development, with examples shown in large antral (A), early antral (B), and primary/secondary (C) stages of development. Lower levels of expression were also observed in the theca interna, particularly in large antral follicles (A and D). Images D–F show sections from the same large antral follicle exposed to primary antibody, primary antibody plus 2 µg/ml BMP-6, and no primary antibody. Note the absence of staining in the absence of primary antibody (F) and the marked decline in staining, particularly in the basal membrana granulosa and theca interna in sections coincubated with BMP-6. Image magnifications: x100, x200, x400, x100, x100, and x100 for A, B, C, D, E, and F, respectively.

Gonadotropin and growth factor responses.
There was no difference between genotypes in steroid production by granulosa or theca cells from small follicles during the initial 48 h of culture, but in cells exposed to the optimum doses of gonadotropins, insulin, and IGF-I LR3 known to induce cellular differentiation (36, 37), induction profiles differed between genotypes with higher levels of production by somatic cells from Fec^B/B animals from 96 h of culture onward (Fig. 5).

Granulosa cells from both genotypes responded to stimulation by FSH in a dose-responsive manner (P < 0.001), with cells from Fec^B/B ewes exhibiting a significantly greater response at doses greater than 1 ng/ml (Fig. 6A; P < 0.01). Granulosa cells from neither genotype responded to stimulation with LH, although estradiol production levels were higher (P < 0.05) in cells from Fec^B/B across the dose range (Fig. 6B). There was no difference between genotypes in proliferative responses of either granulosa or theca cells (data not shown) to either FSH or LH. Granulosa cells from both genotypes cultured in the presence of optimum doses of FSH, insulin, and IGF-I exhibited similar responses to the mitogenic growth factor TGF, with a marked depression in estradiol production (Fig. 6C; P < 0.001) and a concomitant 44–56% increase in cell number (P < 0.01), resulting in no significant interaction between genotype and dose of TGF for either of these parameters. However, there was a significant (P < 0.05) effect of genotype on estradiol production due to the higher levels of production by cells from Fec^B/B animals at the zero dose (Fig. 6C). A converse dose response was obtained when cells were exposed to the differentiative factor IGF-I, in the presence of 10 ng/ml insulin and 10 ng/ml FSH, with a marked stimulation in estradiol production in both genotypes (P < 0.001). In this instance, however, there was a highly significant effect of genotype (P < 0.001) and a significant interaction (P < 0.01), with cells from Fec^B/B follicles exhibiting a greater response to doses greater than 1 ng IGF-I LR3/ml (Fig. 6D). There was no difference between genotypes in the proliferative response of granulosa cells to IGF-I (data not shown).

View larger version (27K): [in this window] [in a new window]   FIG. 6. Effect of FecB mutation on response of granulosa cells to gonadotropins and other growth factors. These data show estradiol production by granulosa cells isolated from small antral follicles of Fec+/+ (open column) and FecBF/F (closed column) animals and cultured under serum-free conditions in the presence of increasing doses of FSH (A), LH (B), TGF (C), and IGF LR3 (D). FSH and LH doses responses were conducted with 10 ng/ml insulin and IGF-I. TGF dose responses were conducted with 10 ng/ml FSH, insulin, and IGF-I. IGF dose responses were conducted with 10 ng/ml insulin and FSH. Values are mean ± SEM, and asterisks denote significant difference between genotypes at the same dose: *, P < 0.05, **, P < 0.01, and ***, P < 0.001. Note that the data have been corrected for cell number.

View larger version (13K): [in this window] [in a new window]   FIG. 7. Effect of FecB mutation on response of theca cells to gonadotropins and other growth factors. These data show androstenedione production by theca cells isolated from small antral follicles of Fec+/+ (open column) and FecBF/F (closed column) animals and cultured under serum-free conditions in the presence of increasing doses of LH (A), TGF (B), and IGF LR3 (C). LH doses responses were conducted with 10 ng/ml insulin and IGF-I. TGF dose responses were conducted with 0.1 ng/ml LH and 10 ng/ml insulin and IGF-I. IGF dose responses were conducted with 0.1 ng/ml LH and 10 ng/ml insulin. Values are mean ± SEM, and asterisks denote significant difference between genotypes at the same dose: *, P < 0.05, **, P < 0.01; and ***, P < 0.001. Note that the data have been corrected for cell number.

View larger version (33K): [in this window] [in a new window]   FIG. 8. Effect of FecB mutation on response of granulosa cells to BMPs. These data show estradiol (A and C) and inhibin A (B and D) production by granulosa cells isolated from small antral follicles of Fec+/+ (open column) and FecBF/F (closed column) animals and cultured under serum-free conditions for 192 h in the presence of increasing doses of BMP. Because there was no significant difference among BMP-2, -4, and -6, data have been pooled within genotype. All dose responses were conducted in the presence of 10 ng/ml FSH and either 0.1 ng/ml (A and B) or 10 ng/ml (C and D) IGF-I LR3. Values are mean ± SEM, and asterisks denote significant difference between genotypes at the same dose: *, P < 0.05, **, P < 0.01, and ***, P < 0.001. Note that the data have been corrected for cell number.

View larger version (21K): [in this window] [in a new window]   FIG. 9. Effect of FecB mutation on response of theca cells to BMPs. These data show cell number (A) and androstenedione production (B) by theca cells isolated from small antral follicles of Fec+/+ (open column) and FecBF/F (closed column) animals and cultured under serum-free conditions for 144 h in the presence of increasing doses of BMP-6. The dose responses were conducted in the presence of 0.1 ng/ml FSH, 10 ng/ml insulin, and IGF-I LR3. Values are mean ± SEM, and asterisks denote significant difference between genotypes at the same dose: *, P < 0.05, **, P < 0.01, and ***, P < 0.001. Note that the androstenedione production data have been corrected for cell number and that the cell number data have been presented as a percentage relative to cell number at the zero dose of BMP-6.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The existence of an interaction between level of gonadotropin stimulation and dose of BMP and IGF in determining level of estradiol secretion by granulosa cells was a major finding of the initial characterization studies. The results presented in Fig. 1 illustrate that, like IGF-I, BMP-6 acts by augmenting FSH-stimulated differentiation because neither IGF-I nor BMP-6 alone is capable of stimulating estradiol secretion under these conditions. Furthermore, the fact that coaddition of low doses of BMP-6 and IGF-I induce an additive augmentation of FSH-stimulated estradiol secretion suggests that each of these stimulatory systems are acting through their separate signaling cascades to influence a common end point, namely the induction of aromatase activity. It is also clear from Fig. 1 that neither BMP-6 nor IGF-I had any effect on the sensitivity of the granulosa cells to FSH stimulation, in which case a shift in the dose-response curve to the left would have been expected. Additional studies with BMP-2 and -4 have also failed to demonstrate any change in granulosa cell sensitivity to FSH (Campbell, B. K., unpublished observations). It is therefore unlikely that BMPs are exerting their effects in sheep through modulating FSH receptor number (13), although this possibility requires more direct experimental evaluation. Finally, the lack of effect of BMP-6 on FSH-stimulated proliferation suggests that unlike IGF-I and insulin (36), BMP-6 does not modulate both proliferation and differentiation in this species.

The results presented in Fig. 2, in which FSH was held constant and dose of BMP and IGF altered across a range of effective doses for local factors, confirm the interactive effect among BMPs (2, 4, and 6) and IGF-I in stimulating induction of estradiol production. The similar nature of the estrogenic responses obtained for BMP-2, -4, and -6 suggests that each of these factors is acting via a common receptor and signaling cascade in ovine granulosa cells. This finding contrasts with that observed in rodents (12) but agrees with the findings of a comparable study in the bovine in which no difference was found among BMP-4, -6, and -7 in terms of their ability to induce secretion of differentiative markers (21). These authors also found that doses of BMP in the range of 2–50 ng/ml were effective and that the magnitude of the response obtained was affected by the dose of IGF-I. However, these results are not directly comparable with those reported in the current paper because these bovine cultures were apparently performed in the absence of FSH (21).

In marked contrast to responses observed in granulosa cells, doses of BMP-2, -4, and -6 greater than 0.5 ng/ml inhibited LH-stimulated thecal androstenedione production, whereas very low doses, ineffective in granulosa cells, stimulated an increase in thecal cell number. Furthermore, there was a difference between BMPs in that BMP-6 was a less potent inhibitor of androstenedione production than BMP-2 and -4 (Fig. 3). The most likely explanation for these apparently contradictory responses of ovarian somatic cells to BMP stimulation is suggested by the results of immunohistochemical analysis of BMP-6 expression in sheep ovary (Fig. 4), in which intense staining is seen in the granulosa cell layer with much fainter, but definite, staining for BMP-6 in the theca cell layer. It is therefore likely that theca cells may be exposed to very low levels of BMP in vivo and that the higher doses observed to be inhibitory in vitro are in fact superphysiological for this cell type. In this case, the observed proliferative response to very low levels of BMP would be consistent with a role for BMP in increasing the steroidogenic potential of a follicle by increasing the number of steroidogenic thecal cells. This interpretation is supported by the fact that total androgen production is increased at low doses of BMP, due to the increase in cell number (Fig. 3C). Thus, we would hypothesize that during the growth and maturation of ovarian follicles, as they move into the gonadotropin-dependent phase (8), higher levels of exposure of granulosa cells to BMP would augment FSH-stimulated estradiol production, whereas lower levels of exposure of theca cells to BMP would in turn increase LH-stimulated androgen precursor supply for estrogen production. The potent inhibitory effect of BMPs on thecal androstenedione production has recently been confirmed by data from the cow using a similar serum-free culture system (42). However, in the cow the BMPs seem to be acting as a more classical local factor in which proliferative and differentiative effects were inversely related, so that proliferative effects were observed only at higher doses of BMP greater than 1 ng/ml. Further work is required to determine whether this difference is due to species or methodological effects.

The demonstration that BMPs are expressed in vivo by sheep ovarian somatic cells was essential if we were to conclude that the responses obtained to these factors in vitro had physiological relevance. The results presented in Fig. 4 support data from other species (15) in showing that BMP-6 is expressed by the sheep oocyte and therefore, like GDF-9 and BMP-15, may be secreted by the oocyte to modulate somatic cell function (43). Unlike reports from rodents (15) and isolated bovine somatic cells (21), we observed very intense staining for BMP-6 in the membrana-granulosa and less intense staining within the theca cell layer of most growing preantral and antral follicles in sheep. The specificity of this BMP-6 protein expression was confirmed by the marked reduction in the intensity of staining in the basal membrana granulosa and thecal cell layer in sections that had been coincubated with rhBMP-6. The staining that remained on the granulosa cells lining the antral cavity may have been nonspecific or due to high local concentrations of endogenous protein in the follicular fluid and around these cells. Similar expression patterns for BMP-6 have also been observed with fixed ovarian tissue from calves and cows and the specificity of this antibody for BMP-6 confirmed (44). Whereas these results support a physiological role for at least one of the BMPs tested in this work in modulating granulosa and theca cell function (see above), analysis of mRNA expression by in situ hybridization is required to confirm the source of the protein expression observed. Although it is likely that the oocyte is a source of BMP-6 expression within the sheep follicle, the high level and widespread distribution of BMP-6 protein expression within the granulosa cell layer of sheep antral follicles suggests that the granulosa cell layer may also secrete this factor in vivo. Conversely, the low level of BMP-6 expression in the theca cell layer of sheep antral follicles is consistent with the idea that for these cells this protein is acting as a paracrine factor derived from the oocyte and/or granulosa cell layer. However, Glister et al. (21) reported BMP-4 and -7 protein expression in isolated bovine theca cells and the confirmation of this hypothesis must await further analysis by in situ hybridization. As part of these expression studies, attempts were also made to examine the pattern of expression of BMP-2 and -4 protein in the ruminant ovary, but in our hands the commercial antibodies currently available for these proteins produced equivocal results (Dugan, K. and R. Webb, unpublished results). The demonstration, however, by Northern analysis that sheep ovary express mRNA for BMP-2, -4, -6, and -7 (3) strongly suggests that these factors also play a physiological role in the controlling ovarian function in these species. More systematic studies are therefore required to examine the effect of stage of follicle maturity and health on the pattern of mRNA and protein expression for components of the BMP system in monovulatory species.

The results of the culture studies on ovarian somatic cells from wild-type and carrier ewes of the FecB mutation confirm the results obtained from the BMP characterization studies and reveal a profound effect of the FecB mutation on both granulosa and thecal cell responsiveness to gonadotropin, IGF, and BMP stimulation in vitro. Induction profiles for estradiol and androstenedione production by granulosa and theca cells, respectively (Fig. 5), showed that the cells did not differ in their steroidogenic potential during the initial periods of culture but that under the influence of optimum doses of gonadotropin and IGF-I, induction profiles diverged so that cells from FecB animals produced two to three times more steroid at the end of culture on a per-cell basis than cells from wild-type animals. Examination of the gonadotropin and IGF-I dose responses (Figs. 6 and 7) in these two cell types revealed that these genotypic differences were evident only at combined FSH/LH and IGF-I doses shown previously to induce steroid production (36, 37), revealing a clear interaction between these two differentiative factors in revealing the action of the mutation in vitro. Similarly, the more recent studies examining the effect of the mutation of BMP responses of granulosa cells (Fig. 8) showed that whereas the genotype effect is evident at maximal doses of IGF-I (10 ng/ml), suboptimal doses of IGF-I (0.1 ng/ml IGF-I) are required to reveal a marked increase in the sensitivity of granulosa cells from FecB mutants to BMP stimulation, in terms of both estradiol and inhibin A production.

View larger version (13K): [in this window] [in a new window]   FIG. 5. Time course of estradiol (top) and androstenedione (bottom) production by granulosa and theca cells, respectively, isolated from small antral follicles of Fec+/+ (closed circles) and FecBF/F (open circles) animals and cultured under serum-free conditions. Granulosa cells were cultured in the presence of 10 ng/ml oFSH, 10 ng/ml insulin, and 10 ng/ml IGF LR3 and theca cells in the presence of 0.1 ng/ml oLH, 10 ng/ml insulin, and 10 ng/ml IGF LR3. Note that levels of production do not differ between genotypes during initial periods of culture and then diverge as the cells differentiate. Values are mean ± SEM, and asterisks denote significant difference from the 48-h time point: **, P < 0.01; ***, P < 0.001.

Whereas differences between genotypes in responses of granulosa cells to stimulation with BMPs, even more profound effects were found in the response of the theca cells. These studies were restricted to the known physiological ligand BMP-6, and the results show a complete reversal to the responses observed in cells from wild-type animals (Figs. 3 and 9), with no proliferative response and a marked augmentation of LH-stimulated androgen production at doses less than 0.5 ng/ml (Fig. 9).

Overall, the results of these culture studies show an increased differentiative response of both granulosa and theca cells to BMP, gonadotropin, and IGF-I stimulation ovarian tissue from FecB animals. Because the FecB mutation is known to be in the intracellular signaling region of the BMPR-1B, then the most likely explanation for the effects observed is that they occur as a consequence of the mutation causing an increased responsiveness of somatic cells to gonadotrophic stimulation through the interactions between BMPs and IGF-I observed in the present paper. The work of Fabre et al. (45), in which wild-type and FecB (Q249R) mutant BMPR-1B were transfected into human kidney cells, both supports and contradicts this hypothesis in that the FecB transfected cells exhibited an increase in basal luciferase activity (relative to the wild type) but failed to respond to stimulation with BMP-4. This latter result is, however, at odds with the findings of cell culture experiments in the same paper and also in the work reported here (Figs. 8 and 9), showing that cells from FecB mutants do respond to BMP-4 and other members of the BMP family in vitro. Because differences in the cellular responsiveness of somatic cells from wild-type and FecB mutants persist in vitro under conditions in which exogenous BMPs are not added, for this hypothesis to be correct, endogenous sources of BMPs or other natural ligands must be present within the cultured cells. From the results of immunohistochemical analysis of ovarian BMP protein expression in ruminants presented in this and other papers (21), this appears to be very likely.

The alternative explanation for the increased responsiveness of somatic cells from FecB mutants to differentiative factors is that, although the cells came from the same size class, the effect of the mutation in inducing precocious follicular maturation meant that the cells were at a different stage of differentiation at the time of isolation. Two pieces of evidence argue against this interpretation. First, although from the same small follicle size class, changes in the follicular hierarchy caused by FecB mutation means that the majority of small follicles dissected from FecB animals were around 1 mm in diameter, whereas the majority of follicles dissected from wild-type animals were around 2 mm in diameter. The most likely explanation for this difference is that the cut-off for the transition from gonadotropin-responsive to gonadotropin-dependent status (8, 50) is altered by the mutation so that the pool of recruitable follicles tends to accumulate at a smaller diameter in FecB mutants. If this interpretation is correct, then we are comparing cells from follicles at a similar differentiative state, and this idea is supported by the fact that the time-course data show no difference in steroidogenic output during the initial periods of culture with similar induction profiles, with the main effect of the mutation being that it allowed cells to respond to differentiative stimuli to achieve a higher maximum level of steroid and inhibin A production. Second, we have shown that dispersion of ovarian somatic cells before culture results in a rapid loss in the mRNA expression of most differentiative markers, including FSH receptor, LH receptor, and aromatase, during the initial 24 h of culture, followed by a rapid sequential mRNA induction phase that precedes the induction of cellular steroidogenic activity (51). Thus, even if the somatic cells were at a more advanced differentiative stage at the time of collection, it is extremely likely that these disruptive effects would have reduced the cells to a common differentiative baseline during the initial period of culture.

In conclusion, the results of the present studies are consistent with a major role for the BMP system in a monovulatory species in modulating proliferative and differentiative responses of both granulosa and theca cells to gonadotrophic stimulation and indicate a significant interaction of BMPs with the IGF system. Culture studies using ovarian tissue from ewes with the FecB mutation showed that the mutation resulted in an increased differentiative response of both granulosa and theca cells to BMP, gonadotropin, and IGF-I stimulation, thus explaining the profound effect of the FecB mutation in inducing precocious maturation of ovarian follicles and hence deregulating the normal follicle selection mechanisms operating in this species.

	Acknowledgments

 
We gratefully acknowledge the technical assistance of Mrs. Catherine Pincott-Allen, Mr. Neil Hollow, and Mrs. Joan Docherty. We thank the National Institute of Arthritis, Metabolism, and Digestive Diseases for the ovine gonadotropins and inhibin, Professor B. Cooke for the androstenedione assay reagents. and Professor N. Groome for the inhibin A assay reagents.

	Footnotes

 
This work was supported by Biotechnology and Biological Science Research Council Research Grant 42/S20230 and European Union Grant CT 92-0232.

The authors have no conflict of interest.

First Published Online January 5, 2006

Abbreviations: BMP, Bone morphogenic protein; BMPR, BMP receptor; Fec^+/+, animals without FecB mutation; GDF, growth differentiation factor; kcells, 1000 cells; o, ovine; rh, recombinant human.

Received May 18, 2005.

Accepted for publication December 22, 2005.

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