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Department of Reproductive and Developmental Sciences, University of Edinburgh (R.B.S., S.G.H., P.L., C.R.H.), Edinburgh, United Kingdom EH3 9ET; and Department of Gene Expression and Development, Roslin Institute (G.M., M.C.), Roslin, United Kingdom EH25 9PS
Address all correspondence and requests for reprints to: Stephen G. Hillier, Ph.D., Department of Reproductive and Developmental Sciences, University of Edinburgh, 37 Chalmers Street, Edinburgh, United Kingdom EH3 9ET. E-mail: s.hillier{at}ed.ac.uk
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Abstract |
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-dihydrotestosterone (DHT;
10–6 M), or FSH plus DHT. Total
cellular RNA was extracted from cells at 6, 12, 24, and 48 h of
treatment for DDRT-PCR analysis, corresponding to an estimated 60%
saturation of the messenger RNA (mRNA) population. Six distinct
complementary DNA clones were obtained that reproduced the DDRT-PCR
profile on a Northern blot of the corresponding RNA samples. Two of
these clones detected transcripts that were strongly down-regulated by
FSH. One corresponded to connective tissue growth factor (CTGF), a
cysteine-rich secreted protein related to platelet-derived growth
factor that is implicated in mitogenesis and angiogenesis, and a second
was identical to lysyl oxidase (LO), a key participant in extracellular
matrix deposition. In detailed expression studies, Northern analysis
revealed a single, approximately 2.5-kb CTGF transcript maximally
suppressed within 3 h of exposure to FSH with or without DHT and
two LO transcripts (
3.8 and 5.2 kb) maximally suppressed at
6 h. DHT alone did not affect CTGF mRNA, but strongly enhanced LO
mRNA relative to the control value. In vivo, CTGF and LO
transcripts were significantly suppressed in GC 48 h after equine
CG injection (10 IU, ip) compared with untreated controls and were
further reduced 12 h after administration of additional 10 IU hCG
to induce luteinization. In situ hybridization confirmed
GC in preantral/early antral follicles as principal sites of CTGF and
LO mRNA expression. We conclude that expression of CTGF and LO mRNAs is
inversely related to GC differentiation. The encoded proteins probably
have roles in the regulation of tissue remodeling and extracellular
matrix formation during early follicular development. |
Introduction |
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Searching for candidate genes involved in this intimate cell dialogue, we examined differences in gene expression caused by exposure of rat granulosa cell cultures to FSH and androgen using differential display RT-PCR (DDRT-PCR) (7). An immature GC culture system was selected to reveal differentiation-related changes under experimentally defined conditions in vitro. Here, we report evidence that connective tissue growth factor (CTGF) and lysyl oxidase (LO) are members of the FSH/androgen-regulated gene repertoire expressed in mammalian granulosa cells. As CTGF is implicated in the regulation of connective tissue synthesis (8, 9) and LO in the formation of ECM (10, 11), both genes are likely to play physiologically significant roles in the regulation of ovarian follicular development.
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Materials and Methods |
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Animals
Immature (21-day-old) female Wistar rats from Charles River Laboratories, Inc. UK (Margate, UK), were kept in a
temperature-controlled room on a 12-h light, 12-h dark cycle and fed
rat chow and water ad libitum. To provide ovaries containing
proliferating but essentially nondifferentiated granulosa cells for
tissue culture (see below), animals were given daily sc injections of
DES (2 mg/day) for 2 days to stimulate preantral/early antral
follicular development. For expression studies in vivo,
gonadotropin-stimulated ovaries were obtained 48 h after ip
injection of eCG (10 IU) or 12 h after ip injection of hCG, given
48 h after injection of 10 IU eCG. Controls received no hormone
treatment.
GC isolation and culture
Ovaries were removed for GC isolation after killing the animals
by asphyxiation with CO2. All handling and
treatment of animals were performed according to guidance issued by the
British Home Office. Follicles were punctured with a 25-gauge
hypodermic needle, and GC were gently expelled into culture medium. The
cells were harvested by centrifugation, resuspended in fresh medium,
and counted in a hemocytometer. Cell viability assessed by trypan blue
staining was 30% or more. For tissue culture studies (cells from
DES-treated animals), 24-well tissue culture grade plastic dishes
(Corning, Inc., Corning, NY) were precoated with donor
calf serum and washed twice with Dulbecco’s PBS before inoculation
with 0.25 ml culture medium containing approximately 2 x
105 viable cells (12). After
overnight preincubation at 37 C in a humidified incubator gassed with
5% CO2 in air, prewarmed culture medium (0.25
ml) containing hormone(s) was added to start the following treatments:
FSH (1 or 10 ng/ml), DHT (1 µM), or FSH plus DHT. Control
cultures contained no hormone. Thirty-six wells were used for
each treatment at each time point. Incubation was performed for 0–48 h
at 37 C. The index response was aromatase activity, measured by
incubating washed cell monolayers for an additional 6 h at 37 C in
0.5 ml medium containing 1 µM testosterone as an
aromatase substrate. This medium was collected and assayed for
estradiol content by RIA (12).
RNA isolation and RNA gel blot analysis
Total RNA was isolated using RNAzol B (Tel-Test,
Friendswood, TX) following the manufacturer’s recommendations. To
obtain RNA from cultured granulosa cell monolayers, RNAzol B (150 µl)
was added to the culture wells after removing spent culture medium; for
freshly isolated granulosa cells, RNAzol was added after collecting the
cells by centrifugation in 1.5-ml Eppendorf (Eppendorf AG,
Hamburg, Germany) tubes. RNA was fractionated by electrophoresis
on 1.0% agarose-formaldehyde gels. Northern hybridization was carried
out according to standard methods using nylon filters and hybridizing
in 7% SDS, 0.5 M sodium phosphate (pH 7) at 65 C.
Posthybridization washes were two washes for 5 min each time in 2
x SSC (standard saline citrate) and two washes for 15 min each time in
0.1 x SSC in presence of 0.1% SDS at 65 C. The probes were
32P-labeled amplicons yielded by DDRT-PCR
analysis of GC RNA (see below), rat cytochrome P450 aromatase
(P450arom) complementary DNA (cDNA; provided by
Dr. J. S. Richards, Baylor College of Medicine, Houston, TX), and
rat 18S ribosomal RNA (rRNA) cDNA (Ambion, Inc., Austin,
TX). The filters were exposed to autoradiographic film (XAR-5,
Eastman Kodak Co., Rochester, NY) for 1–2 days at –70 C
using an intensifying screen.
DDRT-PCR analysis
The source material for DDRT-PCR analysis was total RNA isolated
from cultured GC from DES-treated immature rats incubated for 6, 12,
24, and 48 h with and without FSH, DHT, or FSH plus DHT. DDRT-PCR
was performed essentially as previously described (7) with
minor modifications (13, 14, 15). The DDRT-PCR screen was
performed using 60 separate primer combinations (3 anchor primers and
20 random primers) corresponding to an estimated 60% saturation of the
messenger RNA (mRNA) population. Radiolabeled DDRT-PCR products were
loaded onto a 6% nondenaturing HR-1000 GenomyxLR polyacrylamide gel
(Genomyx, Foster City, CA). Samples were run for 2 h and 15 min at
2700 V (50 C) on a GenomyxLR DNA analyzer (Beckman Coulter, Fullerton, CA). A representative subset of bands
exhibiting a range of hormone response profiles was selected for
further processing, as previously described (13). After
purification (14), cDNAs were PCR reamplified and cloned
into the EcoRI site of pBluescript SK+
(Stratagene Europe, Amsterdam, The Netherlands). The
authenticity of isolated DDRT-PCR candidate cDNAs was confirmed
(15) before expression studies by probing duplicate
Southern-blotted DDRT-PCR products. Sequencing was performed using the
Promega Corp. cycle sequencing kit (Promega Corp. Europe, Southampton, UK) according to the manufacturer’s
instructions, using M13 forward and reverse primers (14, 15). To determine the identity of isolated DDRT-PCR cDNAs, all
sequence data were checked against nonredundant GENBANK/EMBL protein
and nucleotide databases, using the National Center for Biotechnology
Information worldwide web implementations of the BLAST algorithm
(16).
In situ hybridization
Ovaries were fixed in 4% paraformaldehyde in PBS and embedded
in paraffin blocks. After digestion with proteinase K and acetylation,
sections (10 µm) were hybridized (overnight incubation at 55 C) with
complementary RNA (cRNA) probes generated from cDNA templates labeled
with [35S]UTP (Amersham International, Aylesbury, UK) using an RNA transcription kit
(Promega Corp.). Slides were washed in buffers of
decreasing salt concentrations, dehydrated through ethanol gradients,
and processed for liquid emulsion autoradiography (Kodak
NTB-2). After exposure for 3 weeks at 4 C, slides were developed,
counterstained with hematoxylin, and taken for photomicrography. Sense
cRNA probes were used as a control for nonspecific binding.
Data analysis
Northern blots were quantified by electronic autoradiography
(Instant Imager, Packard, Downers Grove, IL) with normalization of mRNA
abundance to 18S rRNA. Data were analyzed using one-way ANOVA with
Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and Northern analysis of
P450arom mRNA (data not shown) confirmed
hormone-responsive gene expression with synergy between FSH and DHT in
the GC culture system from which input RNA was obtained for DDRT-PCR
analysis.
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). Both clones
detected transcripts that were strongly down-regulated by FSH (Fig. 3). One corresponded to nucleotides
934-1340 of full-length rat CTGF cDNA (17), which encodes
a secreted protein implicated in tissue remodeling and angiogenesis
(18). The second was identical to nucleotides 1868–2428
of rat lysyl oxidase cDNA (19), which encodes a key
participant in ECM deposition (20).
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). Within 3 h of exposure to
FSH, the CTGF transcript was strongly suppressed relative to the
control value. The response to FSH was not measurably affected by the
additional presence of DHT. DHT alone had no effect relative to the
control.
GC LO mRNA expression is suppressed by FSH, but enhanced by androgen, in vitro
Freshly isolated GC also expressed an approximately 5.2-kb LO mRNA
transcript and a less abundant approximately 3.8-kb transcript, both of
which declined and then gradually recovered during culture in the
absence of hormones (Fig. 3B). Within 6 h of treatment with FSH in
the presence and absence of DHT, both transcripts were fully
suppressed. Conversely, treatment with DHT alone markedly increased the
expression of both transcripts relative to the control.
Gonadotropin treatment in vivo down-regulates GC CTGF and LO mRNA expression
To determine whether the differentiation-associated changes
observed in vitro reflected physiological events in
vivo, immature female rats were treated with eCG with or without
hCG 48 h later to promote preovulatory follicular development.
Treatment with eCG alone caused approximately 50% (P< 0.01) down-regulation of granulosa cell
CTGF and LO mRNA expression relative to controls. Additional treatment
with hCG to induce luteinization caused further significant
suppression of both mRNAs (Figs. 4 and 5).
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) and LO (Fig. 6E) mRNA signals. The
CTGF signal was more intense than that of LO and was expressed in many
primary and secondary follicles from which LO signaling was absent.
Consistent with the overall lack of follicular maturity in control
animals, P450arom expression was uniformly
negative (Fig. 6I). GC in large antral follicles induced by treatment
with eCG abundantly expressed P450arom mRNA (Fig. 6J), but showed reduced expression of both CTGF (Fig. 6B) and LO (Fig. 6F) mRNA. Additional 12-h treatment with hCG to induce GC luteinization
further suppressed CTGF (Fig. 6C) and LO (Fig. 6G). The CTGF mRNA
signal persisted in the most antrally located granulosa layer and
cumulus GC of luteinizing antral follicles (Fig. 7).
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Discussion |
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Expression of CTGF mRNA in rat ovarian cells has not previously been described, although a recent report (22) demonstrated CTGF mRNA and protein in follicles and corpora lutea of porcine ovary. CTGF protein has also been detected in human follicular fluid (23). The DDRT-PCR product used here to detect ovarian CTGF mRNA expression was a 716-nucleotide sequence corresponding to the region of the rat CTGF cDNA open reading frame (16) that is predicted to encode the whole of domain 4 and the C-terminal half of domain 3. As in other tissues that express CTGF mRNA, only a single approximately 2.5-kb transcript was observable by Northern analysis, consistent with the full-length (2345-bp) CTGF transcript.
CTGF is a member of the connective tissue growth factor/cysteine-rich 61/nephroblastoma-overexpressed (CCN) family of growth factors predicted to have arisen from a common ancestral gene more than 40 million yr ago (see Ref. 9 for review). Other members of the family include Fisp 12 (murine ortholog of human CTGF), human and murine Cyr61, the chicken ortholog of Cyr61, Cef10, and human and Xenopus Nov. With the exception of Nov, these are immediate early genes induced by serum, growth factors, or certain oncogenes. The encoded products are cysteine-rich secretory proteins organized into four distinct motifs. The first contains an insulin-like growth factor-binding domain that is common to the seven known insulin-like growth factor-binding proteins, the second contains a von Willebrand factor type C module thought to be involved in oligomerization, the third is a thrombospondin type I repeat thought to mediate cell attachment and binding to ECM, and the fourth is a C-terminal domain implicated in heparin binding and dimerization (9). Recently, a novel CTGF-like (CTGF-L) gene, structurally related to CTGF but lacking the C-terminal domain, was identified and cloned from human osteoblasts (24). CTGF-L mRNA was also expressed in fibroblasts, heart, testes, and, most abundantly, ovary, but cell localization in the ovary was not reported. In view of their functional properties in other tissues where they are expressed and their pattern of expression during GC differentiation, it seems highly likely that CTGF, CTGF-L, and possibly other members of the CCN growth factor family serve significant paracrine roles in the ovary.
Coexpression of CTGF and LO mRNAs in GC is consistent with connective tissue biosynthesis and ECM deposition occurring hand in hand during follicular development (1, 2). A functional link between the two genes is further suggested by the observation that CTGF can stimulate LO enzyme activity and insoluble collagen formation in human gingival fibroblasts (25). ECM synthesis involves deamination of peptidyl lysine residues to allow covalent inter- or intrachain cross-linking of collagen and elastin fibrils catalyzed by LO (26). The degree of collagen and elastin cross-linking varies between tissues and is constantly altering in the lamina basalis of developing ovarian follicles (1, 27). Here we documented strong LO mRNA expression by GC in preantral and early antral follicles that subsided in response to gonadotropic stimulation in vitro and in vivo. The abundant, approximately 5.2-kb and the rarer, approximately 3.8-kb GC LO mRNA transcripts correspond to similarly sized LO transcripts in rat vascular smooth muscle cells (19, 28). However, the regulation, secretion, and extracellular activities of LO at any cellular site remain poorly understood. LO enzyme activity in rabbit ovarian follicles is reported to be increased after hCG-induced ovulation (29), and LO mRNA expression is up-regulated at the time of ovulation in perch ovary (30); otherwise, there is no scientific literature on ovarian LO. Therefore, it remains to be determined whether LO of GC origin has a particular role in ECM deposition during folliculogenesis, as predicted by the pattern of LO gene expression observed here.
CTGF and LO mRNAs are sequentially repressed in GC under conditions of
culture that promote cellular differentiation in vitro. The
rapid (3-h) suppression of CTGF mRNA upon exposure to FSH is consistent
with the designation of the CTGF gene as immediate-early in
fibroblasts, endothelial cells, vascular smooth muscle cells,
epithelial cells, chondrocytes, and glioblastoma cells (9, 31). However, CTGF mRNA expression at these sites is typically
up-regulated by serum enrichment or exposure to tissue growth factors
such as transforming growth factor-ß (TGFß), platelet-derived
growth factor (PDGF), or basic fibroblast growth factor. It remains to
be determined what effects these or any other growth/differentiation
factors might exert on GC CTGF expression, but FSH is suppressive. LO
mRNA expression in immature GC was also down-regulated by FSH, albeit
less abruptly than CTGF. Again, this contrasts with LO expression
profiles in other tissues, such as lung fibroblasts (32, 33) and rat vascular smooth muscle cells (34, 35),
where PDGF, angiotensin II, or serum enrichment induces LO mRNA. An
exception to this rule is interferon-
, a proinflammatory cytokine
present in aneurysm and arteriosclerotic plaque rupture that
down-regulates LO gene expression in rat aortic smooth muscle cells
(36).
Down-regulation by FSH of CTGF and LO gene expression in GC is presumably mediated by cAMP-mediated postreceptor signaling. Elevation of cAMP levels within target cells by a variety of methods blocks the induction of CTGF by TGFß1 (37, 38). Moreover, TGFß1-induced LO mRNA expression in human lung fibroblasts is inhibited by PGE2 via increased formation of cAMP (33, 39), and cAMP-mediated differentiation of preadipocytes into adipocytes is associated with down-regulation of LO expression (40). As the differentiation-inducing action of FSH on GC is also cAMP mediated (41), it seems safe to conclude that postreceptor signaling via this route explains the associated down-regulation of CTGF and LO gene expression. However, this remains to be confirmed experimentally.
The nonaromatizable androgen DHT directly stimulates GC LO gene expression. A precedent for androgenic regulation of LO expression is the stimulation by testosterone of LO enzymatic activity in bovine aorta smooth muscle cells reported by Bronson et al. (42). Androgen treatment in vivo also reduces elastase expression in rat hearts, probably by enhancement of LO activity (43). GC express androgen receptors through which androgen modulates the expression of FSH-inducible genes such as P450arom. It remains to be determined whether DHT induction of GC LO is androgen receptor mediated. The interaction between FSH and androgen also needs to be clarified. The DDRT-PCR screen predicted that DHT would interact with FSH to increase down-regulation of CTGF and LO mRNA expression relative to FSH alone, which was not borne out by the in vitro expression studies. A comprehensive study of the relative contributions of FSH, LH, sex steroids, and other regulatory factors on GC CTGF and LO gene expression in vivo and in vitro is therefore currently under way.
The pattern of expression of CTGF and LO mRNAs in GC suggests that the encoded proteins might play roles in establishing and/or maintaining early follicular cell phenotypes, similar to the postulated role of CTGF in osteoporosis (17). CTGF mRNA is expressed in many tissues, with kidney (44) and brain (45) previously cited as the sites of highest expression. However, direct comparison of kidney and immature rat ovary by in situ hybridization reveals far higher levels of CTGF mRNA in the latter (Hillier, S. G., et al. unpublished). In the rat ovary, the CTGF gene is switched on at the very earliest stages of follicular development when GC have just begun to proliferate. A similar pattern of expression is observed in pig ovary, where CTGF has been hypothesized to promote ovarian cell growth and blood vessel formation during follicular and luteal development (22). Based on its expression profile in the ovary and biological properties in other tissue systems, it is tempting to suggest that CTGF might contribute to the process of thecal cell recruitment, which is a crucial process in folliculogenesis (46). CTGF is structurally and functionally related to PDGF and vascular endothelial growth factor (47), growth factors that are mitogenic for mesenchymally derived cells in blood, muscle, bone/cartilage, and connective tissue. Thus, a role for CTGF as a thecal cell mitogen is also likely. Thecal cells express PDGFß receptor, through which PDGF activates phosphatidylinositol-3-kinase-Akt/protein kinase B and Ras extracellular signal-regulated kinase-1/2 signaling, leading to thecal cell proliferation and enhanced LH-responsive steroidogenesis (48). CTGF reacts with antiserum produced against PDGF and serves as a ligand for the PDGF receptor (8). However, it is unclear which receptors and/or signaling pathways might be activated by CTGF, because a specific CTGF receptor may also exist (49). GC also express PDGF receptors (50), which presumably transduce the mitogenic action of PDGF on these cells (51). Therefore, CTGF could also be an autocrine GC mitogen, particularly during the FSH-independent preantral phase of follicular development when the CTGF gene is most abundantly expressed.
It remains to be established whether the proteins encoded by CTGF and LO mRNAs follow the same differentiation-related pattern of expression in rat GC, although this could be predicted from the parallel expression of mRNA and protein observed during follicular development in the porcine ovary (22). In situ hybridization analyses reveal both genes to be transcriptionally suppressed during preovulatory follicular development induced by eCG, and more so after luteinization induced by hCG. We note that CTGF mRNA remains highly expressed in some antrally located GC for up to 12 h after injection of eCG. Cells in this layer are intimately involved in the formation of the corpus luteum, acting as foci of connective tissue synthesis and neovascularization after ovulation. If, as seems likely (22), biologically active CTGF is produced at this site, an important physiological role in corpus luteum formation is readily envisaged.
In summary, CTGF and LO are gonadotropin-regulated genes in GC. mRNAs encoding these proteins are abundantly expressed in immature follicles at levels inversely related to GC differentiation. LO is up-regulated by androgen in vitro. Cytodifferentiation induced by FSH in vitro or by eCG in vivo is associated with rapid down-regulation of both CTGF and LO mRNAs. This pattern of gene expression predicts that both encoded products are likely to have biologically important roles in the regulation of ovarian follicular development and function.
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Acknowledgments |
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Footnotes |
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Received July 21, 2000.
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References |
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vß3, promotes
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, CTGF mRNA intensity relative to 18S rRNA
(mean ± SEM) measured by electronic autoradiography.
a, Significantly lower (P < 0.01) than
CON; b, significantly lower (P < 0.01)
than both the CON and eCG treatments.
