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Endocrinology Vol. 143, No. 9 3316-3325
Copyright © 2002 by The Endocrine Society

ARTICLE

FSH and TGF-ß Superfamily Members Regulate Granulosa Cell Connective Tissue Growth Factor Gene Expression in Vitro and in Vivo

Christopher R. Harlow, Lindsay Davidson, Kathleen H. Burns, Changning Yan, Martin M. Matzuk and Stephen G. Hillier

Department of Reproductive and Developmental Sciences, University of Edinburgh, Center for Reproductive Biology (C.R.H., L.D., S.G.H.), Edinburgh, United Kingdom EH3 9ET; and Department of Pathology, Baylor College of Medicine (K.H.B., C.Y., M.M.M.), Houston, Texas 77030

Address all correspondence and requests for reprints to: Christopher R. Harlow, Ph.D., Department of Reproductive and Developmental Sciences, University of Edinburgh, Center for Reproductive Biology, 37 Chalmers Street, Edinburgh, United Kingdom EH3 9ET. E-mail: c.harlow{at}ed.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Connective tissue growth factor (CTGF) is a heparin-binding growth factor implicated in diverse epithelial cell types as a paracrine regulator of mitosis, angiogenesis, cellular taxis, and remodeling of the extracellular matrix. To understand the possible roles of CTGF in the ovarian paracrine system, we studied CTGF gene expression by granulosa cells in relation to their stage of cellular differentiation using both in vitro and in vivo methodologies. Untreated monolayer granulosa cell cultures from immature rats abundantly expressed the approximately 2.5-kb CTGF mRNA transcript (determined by Northern analysis), but had low levels of aromatase activity (an index of granulosa cell differentiation). Treatment for 48 h with FSH (0.1–10 ng/ml) dose-dependently inhibited (>=50%) CTGF mRNA expression, but enhanced aromatase enzyme activity. This in vitro observation of CTGF mRNA down-regulation coinciding with FSH-induced granulosa cell maturation is substantiated by studies of in vivo ovarian CTGF expression in FSHß knockout mice. Northern blot and in situ hybridization analyses demonstrate high levels of CTGF expression in the granulosa cells of preantral follicles blocked from further development by the absence of FSH. The action of FSH (10 ng/ml) was mimicked in vitro by 8-bromo-cAMP (1.0 mM) and was augmented by the additional presence of androgen (1 µM 5{alpha}-dihydrotestosterone), consistent with mediation by intracellular cAMP. Conversely, treatment of granulosa cell cultures with TGFß1 (0.1–10 ng/ml) dose-dependently increased CTGF mRNA levels up to 12-fold at a dose of 10 ng/ml, without affecting aromatase activity. Cotreatment with FSH (0.1–10 ng/ml) dose-dependently suppressed the stimulatory action of TGFß1 (10 ng/ml) on CTGF mRNA, but substantially enhanced aromatase activity beyond levels induced by FSH alone. Importantly, other TGFß superfamily members known to be produced in the ovary (growth/differentiation factor-9 and activin A; 10 ng/ml) stimulated granulosa cell CTGF mRNA in a similar fashion as TGFß1 (10 ng/ml), and this was also inhibited by FSH (10 ng/ml). These data show that granulosa cell CTGF gene expression is inversely related to the stage of granulosa cell differentiation, being directly inhibited by FSH via cAMP-mediated signaling. CTGF mRNA abundance in nondifferentiated granulosa cells is up-regulated in vitro by TGFß1, growth/differentiation factor-9, and activin, suggesting paracrine roles for these growth/differentiation factors in the regulation of CTGF synthesis in mammalian ovaries.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CONNECTIVE tissue growth factor (CTGF) is a member of the CTGF/cysteine-rich 61/nephroblastoma overexpressed gene family that mediates TGFß-induced regulation of connective tissue synthesis by a variety of cell types, including fibroblasts (1), chondrocytes (2), osteoblasts (3), vascular smooth muscle cells (4), and renal mesangial cells (5). We have previously shown abundant expression of CTGF mRNA in granulosa cells of preantral and early antral follicles (6). Others have shown CTGF mRNA expression in granulosa and theca of the porcine ovary (7). Intraovarian functions of CTGF remain to be proved, but its involvement in mitosis, angiogenesis, chemotaxis, mototaxis, and cell adhesion to the extra-cellular matrix (ECM) in other cell systems and the pattern of gene expression in granulosa cells predict similar roles for this growth factor in the ovarian paracrine system (8). CTGF is a novel ligand for integrin receptors (9). Moreover, the discovery of the multiligand low density lipoprotein receptor-related protein/{alpha}2-macrogloglobulin receptor as a candidate CTGF receptor (10) highlights the potential biological significance of CTGF in the ovary, as this receptor has been previously identified in both rat (11) and human (12) granulosa cells.

Follicular growth and development are dependent on finely controlled paracrine interactions among the oocyte, granulosa cells, and thecal cells, orchestrated by serum factors, including gonadotropins, and the surrounding ECM (13). In nongonadal epithelial cell types CTGF expression is up-regulated by TGFß1 (1, 2, 3, 4, 5) and down-regulated by factors that promote cytodifferentiation through cAMP-mediated postreceptor signaling (1, 14). Our initial characterization of granulosa cell CTGF mRNA expression (6) showed abundant gene expression in predifferentiated cells, with down-regulation upon exposure to FSH in vitro and in vivo. The FSHß knockout mouse is a useful model for studying the role of FSH in vivo. These mice are infertile due to a block of folliculogenesis before antrum formation (15). Despite the lack of FSH, these follicles develop a well organized thecal cell layer, but the granulosa cells fail to differentiate and do not express cytochrome P450 aromatase mRNA (16).

In the present study we sought further to characterize the inhibitory action of FSH and determine whether TGFß1 and/or other members of the TGFß superfamily of growth/differentiation factors (GDFs) of ovarian origin might be involved in the paracrine regulation of granulosa cell CTGF expression. We used the estrogen-treated immature rat model as a source of undifferentiated granulosa cells for in vitro studies, and the FSHß knockout mouse to investigate the role of FSH in regulating CTGF expression in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Hormones and tissue culture reagents
Diethylstilbestrol, 5{alpha}-dihydrotestosterone (DHT), testosterone, 8-bromo-cAMP (8-br-cAMP), recombinant human (rh) TGFß1, and BSA (fraction V) were obtained from Sigma (Poole, UK). rhFSH (3860 IU/mg) was donated by Dr. C. Howles (Serono Laboratories, Inc, Welwyn Garden City, UK). The recombinant GDF-9 protein was produced as previously described (17). Briefly, CHO cells carrying the full-length mouse GDF-9 cDNA in the pHTop expression vector (a gift from Genetics Institute, Cambridge, MA) were incubated for 48 h in Opti-MEM reduced serum collection medium. The media were harvested, and GDF-9 protein levels were quantified by SDS-PAGE with subsequent immunoblotting using purified recombinant GDF-9 derived from bacteria as a standard. Recombinant human activin A was donated by Dr. T. Woodruff (Northwestern University, Evanston, IL). Culture medium was medium 199 with 25 mM HEPES, supplemented with 2 mM L-glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin (all supplied by Life Technologies, Inc., Paisley, UK), and 0.1% (wt/vol) BSA. Donor calf serum and Dulbecco’s PBS were purchased from Life Technologies, Inc.

Animals
Twenty-one-day-old female Wistar rats (Charles River Laboratories, Inc., Margate, UK) were housed under temperature-controlled conditions on a 12-h light, 12-h dark cycle and were fed rat chow ad libitum. The handling and treatment of the animals was according to the Animals (Scientific Procedures) Act, 1986. Proliferating, but essentially undifferentiated, granulosa cells were induced by giving two daily sc injections of diethylstilbestrol (2 mg/d) in ethanol/propylene glycol (5:95) to stimulate preantral/early antral follicular development.

Mice were maintained as described in the NIH Guide for the Care and Use of Laboratory Animals. Generation of mice carrying the FSHß null allele (Fshbm1, Fshbtm1Zuk) and Southern blot genotyping have been described previously (15). Ovaries were collected from adult C57BL/6/129SvEv (hybrid strain) wild-type (+/+), Fshbtm1Zuk heterozygote (Fshb+/-), and Fshbtm1Zuk/Fshbtm1Zuk (Fshb-/-) mice (6–16 wk of age) for RNA isolation or in situ hybridization.

Granulosa cell isolation and culture
Animals were killed by asphyxiation with CO2, and the ovaries were removed. Granulosa cells were isolated by puncturing follicles with a 25-gauge hypodermic needle and gently expelling the cells into medium. Pooled cells were centrifuged and resuspended in fresh medium, and their viability was assessed by counting a trypan blue-stained preparation in a hemocytometer. Cell viability was 25–30%. Tissue culture grade 24-well plastic dishes (Corning, Inc., Corning, NY) were precoated with 0.25 ml donor calf serum and washed twice with Dulbecco’s PBS (0.5 ml) before being inoculated with 0.25 ml culture medium containing 1 x 105 viable cells. After overnight preincubation at 37 C in a humidified atmosphere containing 5% CO2 in air, 0.25 ml prewarmed medium containing hormone treatments was added (24–36 wells/treatment), and the incubation was continued for 6 or 48 h. Aromatase activity was measured in a separate group of four culture wells after the incubation by washing the cell monolayers twice with prewarmed Dulbecco’s PBS and adding fresh medium containing 1 µM testosterone as substrate. This medium was collected after an additional 3-h incubation and was assayed for estradiol by RIA (18). Aromatase activity data are presented as picomoles of estradiol per 106 cells per hour.

RNA isolation and Northern blot analysis
Total RNA was isolated from rat granulosa cell monolayers using RNAzol B (Tel-Test, Friendswood, TX), with the following modification of the manufacturer’s instructions. Cell monolayers were blotted dry, and 0.1 ml RNAzol B was added to each well. The lysates from 12 wells were pooled and mixed with 0.2 ml chloroform. This mixture was added to a Phase Lock Gel tube (Eppendorf, Hamburg, Germany), and the aqueous phase was separated by centrifugation according to the manufacturer’s instruction.

Total RNA (5 µg) was size-fractionated by electrophoresis on a 1% agarose-formaldehyde denaturing gel for 3 h at 80 V and visualized with ethidium bromide. The RNA was transferred overnight to a nylon membrane (Hybond-N, Amersham Pharmacia Biotech, Little Chalfont, UK), which was then baked for 2 h at 80 C. Rat CTGF transcripts were detected by probing with a cDNA clone corresponding to nucleotides 934-1340 of the full-length rat CTGF cDNA (19) (GenBank accession no. AF 120275), as described previously (6). Northern blot hybridization was performed using standard methods. Hybridization was in Ultrahyb buffer (Ambion, Inc., Huntingdon, UK) at 42 C using 32P-labeled probes (Redivue, Amersham Pharmacia Biotech). Posthybridization washes were two 5-min washes in 2x standard saline citrate and two 15-min washes in 0.1x standard saline citrate, each with 0.1% sodium dodecyl sulfate at 42 C. Hybridization was quantified by electronic autoradiography using an Instant Imager (Packard, Downers Grove, IL) and exposed to autoradiographic film (XAR-5, Eastman Kodak Co., Rochester, NY) for 2–24 h at -70 C. After stripping, blots were reprobed with a rat 18S rRNA cDNA probe (donated by Dr. G. Scobie, Medical Research Council Human Reproductive Sciences Unit, Edinburgh, UK) to allow correction for gel loading and transfer. Northern analysis data are presented relative to unstimulated control values.

Total RNA was isolated from pooled ovaries of 8–16 mice by acid guanidinium thiocyanate-phenol-chloroform extraction using the RNA STAT-60 reagent (Leedo Medical Laboratories, Houston, TX). Fifteen micrograms of each RNA sample were used for electrophoresis and transfer onto a nylon membrane. [{alpha}-32P]Deoxy-ATP-radiolabeled CTGF cDNA probe was synthesized using the Strip-EZ kit (Ambion, Inc., Austin, TX). Autoradiography and phosphorimaging allowed for visualization and quantification of probe hybridization, respectively. Phosphorimaging plates were scanned and analyzed using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). A background level for the blot was determined and subtracted. The membrane was then stripped and reprobed for glyceraldehyde 3-phosphate dehydrogenase, and phosphorimaging of the glyceraldehyde 3-phosphate dehydrogenase signal allowed us to correct each lane for variations in RNA loading.

In situ hybridization
In situ hybridization was performed as previously described (16, 17). Briefly, [{alpha}-35S]UTP-labeled sense and antisense riboprobes were transcribed from CTGF cDNA using T7 and T3 polymerases (Promega Corp., Madison, WI). Paraffin-embedded ovaries were cut into 5-µm sections, dewaxed, fixed, hybridized, and washed. Signal was detected by autoradiography using NTB-2 emulsion (Eastman Kodak Co.). Hematoxylin counterstaining allowed for correlation of the hybridization with granulosa cells within the ovary.

Data analysis
Data were analyzed by one-way ANOVA, using a t test to identify significant differences between treatments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dose-dependent inhibition of CTGF mRNA expression by FSH
Northern analysis showed dose-dependent inhibition of CTGF mRNA by FSH after 48-h incubation, with 10 ng/ml FSH causing a reproducible reduction in expression to approximately 50% of the control value (Fig. 1Go, A and B). By contrast, FSH (Fig. 1CGo) dose-dependently enhanced aromatase activity, an index of granulosa cell differentiation.



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  		Figure 1. Dose-dependent effects of FSH on rat granulosa cell CTGF mRNA (A and B) and aromatase activity (C) after 48-h culture. Controls (Con) received no treatment. A, Autoradiograph from a typical Northern analysis showing dose-dependent inhibition of the approximately 2.5-kb CTGF transcript compared with the signal for 18S rRNA (18S). B, Composite data from five separate experiments. Bars indicate the mean ± SEM CTGF mRNA intensity relative to 18S rRNA measured by electronic autoradiography; all data were normalized to the control value. ANOVA showed a significant effect of treatment (F = 6.54; P = 0.015). a, P < 0.05 compared with controls. C, Aromatase activity measured during a 3-h test period at the end of the experiment. Bars indicate the mean ± SEM levels in culture medium in four separate experiments. ANOVA showed a significant effect of treatment (F = 11.48; P = 0.001). a, P < 0.05; b, P < 0.01 (compared with controls).

 
Intermediary role of cAMP in suppression of CTGF mRNA
The cAMP analog 8br-cAMP (1.0 mM) also significantly suppressed CTGF mRNA levels (Fig. 2Go, A and B). DHT (1 µM), a nonaromatizable androgen that enhances FSH-stimulated cAMP production, augmented the inhibitory effect of FSH (10 ng/ml) on CTGF mRNA levels, which fell to 10–20% of the control value (Fig. 2Go, A and B). This was in contrast to the effect of DHT on FSH-stimulated aromatase activity, which increased 20-fold (Fig. 2CGo). DHT alone also significantly enhanced CTGF mRNA without affecting aromatase activity (Fig. 2Go, A–C).



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  		Figure 2. Actions of FSH (10 ng/ml), 8-br-cAMP (cAMP; 1 mM), and DHT (1 µM) on rat granulosa cell CTGF mRNA (A and B) and aromatase activity (C) after 48-h culture. Controls (Con) received no treatment. A, Autoradiograph from a typical Northern analysis showing CTGF mRNA compared with the signal for 18S rRNA (18S). B, Composite data from three separate experiments. Bars indicate the mean ± SEM CTGF mRNA intensity relative to 18S rRNA measured by electronic autoradiography; all data were normalized to the control value. ANOVA showed a significant effect of treatment (F = 24.5; P < 0.001). a, P < 0.05 compared with controls; b, P < 0.01 compared with FSH; c, P < 0.01 compared with DHT; d, P < 0.01 compared with cAMP. C, Aromatase activity measured during a 3-h test period at the end of the experiment. Bars indicate the mean ± SEM levels in culture medium in three separate experiments. ANOVA showed a significant effect of treatment (F = 391.7; P < 0.001). a, P < 0.05 compared with controls; b, P < 0.01 compared with FSH.

 
Dose-dependent enhancement of CTGF mRNA expression by TGFß1
TGFß1 increased CTGF mRNA expression in a dose- dependent manner, with mean levels increasing 12-fold at a dose of 10 ng/ml (Fig. 3Go, A and B). We observed no effect of TGFß1 on aromatase activity (data not shown).



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  		Figure 3. Dose-dependent effects of TGFß1 on rat granulosa cell CTGF mRNA. Controls (Con) received no treatment. A, Autoradiograph from typical Northern analysis showing CTGF mRNA compared with the signal for 18S rRNA (18S). B, Composite data from five separate experiments. Bars indicate the mean ± SEM CTGF mRNA intensity relative to 18S rRNA measured by electronic autoradiography; all data were normalized to the control value. ANOVA showed a trend, although not significant, due to effect of treatment (F = 2.81; P = 0.073). a, P < 0.01 compared with controls.

 
Dose-dependent inhibition of TGFß1-stimulated CTGF mRNA by FSH
In 48-h incubations FSH dose-dependently suppressed the stimulation of CTGF mRNA levels by TGFß1, with 10 ng/ml FSH reducing CTGF mRNA to 12% of the levels stimulated by TGFß1 alone (Fig. 4Go, A and B). This inhibitory effect of FSH was in contrast to a dramatic FSH-dependent increase in aromatase activity, with 100-fold stimulation by 10 ng/ml FSH in the presence of TGFß1 (Fig. 4CGo).



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  		Figure 4. Dose-dependent effects of FSH on TGFß1-stimulated rat granulosa cell CTGF mRNA (A and B). Controls (Con) received no treatment. A, Autoradiograph from typical Northern analysis showing CTGF mRNA compared with the signal for 18S rRNA (18S). B, Composite data from three separate experiments. Bars indicate the mean ± SEM CTGF mRNA intensity relative to 18S rRNA measured by electronic autoradiography; all data were normalized to the control value. ANOVA showed a significant effect of treatment (F = 0.017; P = 0.017). a, P < 0.05 compared with controls; b, P < 0.05 compared with TGFß1 alone. C, Aromatase activity measured during a 3-h test period at the end of the experiment. Bars indicate the mean ± SEM levels in culture medium in four separate experiments. Compare aromatase levels in response to FSH alone (Fig. 2CGo). ANOVA showed a significant effect of treatment (F = 44.5; P < 0.001). a, P < 0.05; b, P < 0.01 (compared with controls).

 
Time-dependent effects of FSH and TGFß1 on CTGF mRNA
To evaluate changes in CTGF mRNA expression with time, the effects of FSH and TGFß1 at fixed doses (10 ng/ml) on CTGF mRNA were compared at 6 and 48 h. Control, FSH, and TGFß1 CTGF mRNA levels increased 6- to 10-fold from 6 to 48 h, but the signal was suppressed at both time points by treatment with FSH plus TGFß1 (Fig. 5Go, A and B). Inhibition, relative to control, by FSH alone and stimulation by TGFß1 alone were measurable at both 6 and 48 h. Combined treatment with FSH suppressed stimulation by TGFß1, relative to TGFß1 alone, at both time points (Fig. 5Go, A and B).



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  		Figure 5. Effects of TGFß1 (10 ng/ml) and FSH (10 ng/ml) on rat granulosa cell CTGF mRNA after 6- and 48-h treatments. Controls (Con) received no treatment. A, Autoradiograph from typical Northern analysis showing CTGF mRNA compared with the signal for 18S rRNA (18S). B, Composite data from five separate experiments. Bars indicate the mean ± SEM CTGF mRNA intensity relative to 18S rRNA measured by electronic autoradiography; all data were normalized to the control value for 6 h. ANOVA showed a significant effect of treatment (F = 4.37; P = 0.004). a, P < 0.05 compared with 6 h control; b, P < 0.01 compared with 48 h control; c, P < 0.01 compared with 6 h TGFß1; d, P < 0.05 compared with 48 h control; e, P < 0.05 compared with 6 h TGFß1; f, P < 0.05 compared with 48 h TGFß1; g, P < 0.05 compared with 48 h FSH.

 
Comparison of the effects of TGFß1, GDF-9, and activin A on CTGF mRNA
To compare the effects of different TGFß superfamily members on CTGF mRNA expression, granulosa cells were incubated for 6 and 48 h in the presence of 10 ng/ml TGFß1, GDF-9, or activin A in the absence and presence FSH. Stimulation of CTGF mRNA levels occurred at both time points, but was greater at 48 h, and occurred in all three cases, with a trend toward relative potency of activin A > GDF-9 > TGFß1 (Figs. 6Go and 7Go, A and B). The presence of FSH markedly suppressed these effects (Figs. 6Go and 7Go, A and B) while increasing aromatase activity (Fig. 7CGo).



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  		Figure 6. Comparison of the effects of TGFß1, GDF-9, and activin A (Act; all at 10 ng/ml) in the presence or absence of FSH (10 ng/ml) on rat granulosa cell CTGF mRNA after 6- and 48-h treatments. Autoradiograph from typical Northern analysis showing CTGF mRNA compared with the signal for 18S rRNA (18S).

 


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  		Figure 7. A, Composite data from three separate experiments comparing the effects of TGFß1, GDF-9, and activin A (Act; all at 10 ng/ml) in the presence or absence of FSH (10 ng/ml) on rat granulosa cell CTGF mRNA after 6 h of treatment. Bars indicate the mean ± SEM CTGF mRNA intensity relative to 18S rRNA measured by electronic autoradiography in three experiments; all data were normalized to the control value. ANOVA showed a significant effect of treatment (F = 6.72; P = 0.001). a, P < 0.05 compared with controls. B, The same as A, but continued for 48 h, with data normalized to the control value for 48 h. ANOVA showed a significant effect of treatment (F = 15.65; P < 0.0001). a, P < 0.05 compared with controls; b, P < 0.05 compared with corresponding treatment without FSH. C, Aromatase activity measured after 48 h during a 3-h test period at the end of the experiment. Bars indicate the mean ± SEM levels in culture medium in three separate experiments. ANOVA showed a significant effect of treatment (F = 109.5; P < 0.0001). a, P < 0.05 compared with FSH; b, P < 0.05 compared with FSH plus TGFß1; c, P < 0.05 compared with FSH plus GDF-9.

 
CTGF mRNA expression in the FSHß knockout mouse
To further test our model that the FSH response mediates CTGF down-regulation, we investigated the in vivo expression of CTGF mRNA in the absence of FSH using the FSHß knockout mouse model. Northern blot analysis of total ovarian RNA indicates that there is 4-fold up-regulation of CTGF mRNA in mice lacking FSH compared with wild-type mice and Fshb heterozygote littermates (Fig. 8AGo). In situ hybridization experiments indicate that the main source of CTGF expression in both wild-type and FSHß knockout mice is the granulosa cell population in multilayered, preantral follicles. (Fig. 8Go, B–G). There was no hybridization signal detected above background in granulosa cells of preovulatory follicles or in luteinized cells in wild-type ovaries (Fig. 8Go, B–G).



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  		Figure 8. A, Northern blot analysis of CTGF mRNA in ovaries of wild-type (WT), FSH heterozygote (Fshb+/-), and FSH knockout (Fshb-/-) mice. B–G, In situ hybridization analysis of ovarian CTGF expression. B and C, In WT ovaries, the predominant source of CTGF expression is the granulosa cells of multilayered, preantral follicles (asterisks). A representative section is shown at low power magnification in brightfield (B) and darkfield (C) to demonstrate the histology and highlight the hybridization signal, respectively. Probe hybridization is essentially excluded from luteinized cells (CL; B–E) and granulosa cells of small follicles (arrows; B and C) as well as those of large antral follicles (arrows; D and E). F and G, In the absence of FSH, a high level of CTGF expression is seen in the granulosa cells of multilayered preantral follicles that are blocked from further stages of follicular development. Negative control sections probed with sense riboprobes showed no defined pattern of probe hybridization (data not shown).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have further characterized the endocrine and paracrine regulation of CTGF gene expression in the rat ovary and demonstrated that this gene may be tightly controlled by interactions between FSH and TGFß superfamily members. We have also demonstrated the presence of CTGF mRNA for the first time in the mouse ovary and observed that the expression of this gene is restricted to multilayered preantral follicles and is overexpressed in the FSHß knockout mouse.

In our previous study we identified CTGF mRNA expression in rat granulosa cells and demonstrated that FSH down-regulates the expression of CTGF in vitro (6). CTGF mRNA was also suppressed in vivo after exogenous treatment of immature rats with equine CG or equine CG plus hCG (6) We have confirmed the inhibitory role of FSH in the mouse ovary. The high expression of CTGF mRNA in the FSHß knockout mouse, in which there is a block in follicular development at the multilayered preantral follicle stage (15), highlights the importance of CTGF at this stage of development.

FSH acts via its receptor, as the primary regulator of granulosa cell differentiation, to activate adenylate cyclase and increase intracellular cAMP. Steroidogenesis (e.g. estradiol and progesterone synthesis) (20, 21) and induction of LH receptors (22) are mediated in this fashion. We can now add the control of CTGF, a gene involved in ECM remodeling in many other tissues (1, 2, 3, 4, 5), to the repertoire of FSH-regulated genes in the ovary, although in this case FSH, through elevation of cAMP, serves to down-regulate CTGF expression. cAMP also down-regulates CTGF expression in other cell systems, such as rat kidney fibroblasts (1, 13). FSH receptor mRNA expression in human follicles is absent in primordial follicles, is intermittently found in primary and two-layered follicles, and is ubiquitous in multilayered follicles (23), suggesting possible developmental regulation of CTGF by FSH.

Androgens are known to interact with FSH to stimulate a wide variety of differentiation processes, including steroid biosynthesis (21, 24), LH receptor induction (25), and carbohydrate metabolism (26). In the presence of FSH we observed further marked down-regulation of CTGF expression by the nonaromatizable androgen DHT, in direct contrast to the effects on aromatase activity, indicating roles for androgens in the regulation of this gene in the ovary. In primates, androgen, through binding to the androgen receptor, stimulates FSH receptor expression, suggesting a mechanism for the amplifying effects of androgen on FSH-stimulated follicular growth and steroidogenesis (27). Although we have no direct evidence of androgen receptor involvement in CTGF regulation, it seems likely that a similar mechanism is operating. However, DHT synergized with 8-br-cAMP to down-regulate CTGF expression, suggesting that androgen may also act downstream of the FSH receptor to suppress cAMP catabolism, as it does in the stimulation of steroidogenesis (28).

We were interested to note that DHT alone had a significant stimulatory effect on CTGF expression. The action of androgen on CTGF has not been examined in other cell systems, although we have observed stimulation by DHT of another gene involved in connective tissue biosynthesis, lysyl oxidase (the encoded enzyme catalyzes the cross-linking of collagen and elastin monomers to form insoluble ECM) (6). Androgens in bovine aorta smooth muscle cells in vitro (29) and whole mouse cervix in vivo (30) also enhance lysyl oxidase enzyme activity.

TGFß1 mRNA is expressed predominantly in thecal/interstitial cells of immature rat preantral follicles (31), and immunolocalization studies demonstrate the presence of intense staining for TGFß1 at the time of antrum formation (32). We found that TGFß1 dose-dependently increased CTGF mRNA in rat granulosa cells, in line with the observations of TGFß1-induced CTGF regulation in human foreskin fibroblasts (1), NRK fibroblasts (14), and rat osteoblasts (3). As cAMP is able to block TGFß1-stimulated CTGF (1, 3, 14) or downstream ECM events such as collagen synthesis (1), we believe that the dose-dependent inhibition of TGFß1-stimulated CTGF mRNA by FSH is probably mediated by cAMP. Paradoxically, TGFß1 and FSH combinations consistently reduced CTGF mRNA to below the level with FSH alone. However, this could be explained by the ability of TGFß1 to increase FSH receptor expression (33, 34).

The effects of FSH and TGFß1 on CTGF mRNA were observed within 6 h, similar to our previous finding of down-regulation by FSH at 3 h (6) and consistent with the designation of CTGF as an immediate-early gene in fibroblasts, endothelial cells, chondrocytes, osteoblasts, and glioblastoma cells (8). However, the effects of FSH and TGFß1, alone and in combination, increase after 48-h exposure, suggesting stability of the mRNA. Stabilization of CTGF mRNA has been attributed to activation of the kinin B1 receptor (35). Furthermore, serum stimulation increases CTGF mRNA due mainly to transcriptional activation (36).

The actions of FSH on granulosa cells are subject to paracrine modulation, not only by growth factors such as TGFß originating from thecal cells (37), but also by GDF-9 secreted by the oocyte (38, 39). Activin of granulosa cell origin can also modulate FSH action in either an autocrine manner [e.g. cytochrome P450 aromatase activity (40), mitogenic activity (41), FSH and LH receptor mRNA (42), and 17ß-hydroxy-steroid dehydrogenase type 1 (43)] or indirectly by inhibiting LH-induced thecal androgen production (44). Moreover, the expression of FSH receptor mRNA is enhanced by activin A in immature rat granulosa cells (45), and activin is believed to be synthesized preferentially over inhibin at the earliest stages of follicular development (46). Our finding that all three of these TGFß superfamily members enhance CTGF mRNA points to a multifactorial regulation of CTGF activity by the oocyte, theca, and granulosa (Fig. 9Go). The interaction with FSH adds a further dimension to an apparently complex regulatory system and implicates CTGF regulation in the remodeling process of the developing follicle. It is tempting to speculate that the high levels of CTGF mRNA observed in the FSHß knockout mouse result from actions of GDF-9 and/or activin and TGFß released from inhibitory modulation by FSH.



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  		Figure 9. A working hypothesis of FSH and TGFß superfamily actions in the developing follicle. A, In the primary follicle, in the absence of FSH stimulation, GDF-9 and activin stimulate CTGF mRNA expression in granulosa cells. We suggest that the secreted CTGF recruits stromal cells and blood vessels to form the thecal cell layer and orchestrates ECM deposition in the developing basement membrane. B, The multilayered granulosa cells of the preantral follicle have relatively few FSH receptors and are exposed to low levels of FSH. GDF-9, activin, and TGFß are therefore able to stimulate high expression of CTGF mRNA, and the secreted CTGF could coordinate thecal cell proliferation and differentiation as well as formation of the antral cavity. C, In the antral follicle, higher FSH receptor availability and higher FSH levels would serve to switch the effects of activin and TGFß from stimulatory to inhibitory, resulting in reduced CTGF mRNA expression. GDF-9 may have less effect on mural granulosa cells due to gradient effects, as the oocyte is now spatially removed from the mural granulosa cells.

 
The dynamic nature of the mammalian ovarian architecture requires cyclical remodeling of the ECM. Considerable literature is now available on the occurrence and regulation of factors responsible for breakdown of the ovarian ECM. Matrix metalloproteinases and their inhibitors are key components in the breakdown of collagen in the follicular basement membrane, theca interna and externa, tunica albuginea, and surface epithelial basement membrane (47). However, little is known about the factors that regulate the deposition of ECM components, such as collagen and elastin, in these compartments of the ovary, an equally important element of the remodeling process. CTGF has been implicated as a factor regulating collagen deposition in fibroblasts (48), and our observations provide insight into possible mechanisms controlling these events in the ovary.

In situ hybridization analysis in the rat ovary reveals abundant expression of CTGF mRNA in preantral/early antral follicles of immature animals, which is switched off in large antral follicles after eCG treatment at the point when P450AROM expression is maximal (6). In the pig ovary, where a similar pattern of CTGF expression is observed, CTGF is proposed to promote cell growth and angiogenesis during follicular and luteal development (7). Together with the in vitro evidence of a negative correlation between aromatase activity and CTGF mRNA expression, we conclude that differentiation of mature follicles is associated with a down-regulation of the genes involved in maintaining immature, rapidly proliferating, follicular cell phenotypes. A similar regulation is observed in osteoblasts, in which CTGF expression is highest in actively proliferating cells and declines as mineralization becomes evident (19).

We await confirmation of whether CTGF protein expression follows the same pattern as the mRNA in the rat ovary, but this could be predicted from the coexpression of message and protein observed in the pig ovary (7). On the basis of the ovarian expression studies, particularly the presence of CTGF at the earliest stages of follicular development and the regulation of expression by GDF-9, together with the preponderance of activin over inhibin and the known roles of CTGF in ECM remodeling, we propose that CTGF may be a factor involved in transducing the oocyte-derived GDF-9 signal to promote basal lamina development and thecal cell recruitment (Fig. 9AGo). Furthermore, the up-regulation of CTGF by TGFß1 and activin and the attenuation by FSH are indicative of communication between thecal cells and granulosa cells in multilayered preantral follicles, which may regulate antral follicle formation. As a working hypothesis, we propose that in the preantral follicle with low levels of FSH and low FSH receptor availability, the stimulatory actions of TGFß, activin, and GDF-9 on CTGF mRNA expression predominate (Fig. 9BGo). However, in the antral follicle, higher FSH and FSH receptor availability lead to these TGFß superfamily members enhancing the inhibitory effects of FSH on CTGF mRNA expression (Fig. 9CGo).

In summary, CTGF is expressed in granulosa cells in immature rat follicles and is subject to regulation at endocrine, paracrine, and autocrine levels by FSH and TGFß superfamily members originating from the oocyte, thecal cells and granulosa compartment itself. Although TGFß1, GDF-9, and activin are stimulatory, FSH, by raising cAMP levels, is inhibitory and antagonizes the stimulatory effects of TGFß superfamily members. Expression patterns and mode of regulation of CTGF mRNA predict that the encoded protein will have crucial roles in tissue reorganization and ECM deposition during follicular development.


   Acknowledgments
 


   Footnotes
 
This work was supported by Medical Research Council Program Grant 0000066 (to S.G.H.).

Abbreviations: 8-br-cAMP, 8-Bromo-cAMP; CTGF, connective tissue growth factor; DHT, 5{alpha}-dihydrotestosterone; ECM, extracellular matrix; GDF, growth/differentiation factor; rh, recombinant human.

Received December 10, 2001.

Accepted for publication May 15, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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