The Cyr61 and CTGF are members of the CCN family of proteins, which also consists of nephroblastoma overexpressed (Nov/CCN3), Elm-1/WISP-1/CCN4, Cop-1/WISP-2/CCN5, and WISP-3/CCN6. This gene is downstream of Hippo signaling.
Bradham et al. (1991) described a new mitogen produced by human umbilical vein endothelial cells, which they termed
Connective Tissue Growth Factor. The protein, related to platelet-derived growth factor, was predicted
from its cDNA to be a 349-amino acid, 38-kD cysteine-rich secreted protein. Sequence comparison revealed that CTGF belongs to a group known as the immediate-early genes,
which are expressed after induction by growth factors or certain oncogenes . The immediate-early genes
have significant sequence homology to the insulin-like growth factor-binding proteins (IGFBPs) and contain the
conserved N-terminal IGFBP motif. CTGF shares 28 to 38% amino acid identity with IGFBPs
1-6. Kim et al. (1997) proposed that IGFBP7, CTGF , NOV , and IGFBP10 constitute a subfamily of
low-affinity IGFBPs. CTGF-L as well contains an IGF binding domain, a von Willebrand
Factor type C motif, and a thrombospondin type I repeat. However, unlike CTGF, CTGF-L lacks the C-terminal domain
implicated in dimerization and heparin binding.
This gene is in the Hippo pathway.//////Injury-induced ctgfa directs glial bridging and spinal cord regeneration in zebrafish. Mokalled MH et al. (2016) Unlike mammals, zebrafish efficiently regenerate functional nervous system tissue after major spinal cord injury. Whereas glial scarring presents a roadblock for mammalian spinal cord repair, glial cells in zebrafish form a bridge across severed spinal cord tissue and facilitate regeneration. We performed a genome-wide profiling screen for secreted factors that are up-regulated during zebrafish spinal cord regeneration. We found that connective tissue growth factor a (ctgfa) is induced in and around glial cells that participate in initial bridging events. Mutations in ctgfa disrupted spinal cord repair, and transgenic ctgfa overexpression or local delivery of human CTGF recombinant protein accelerated bridging and functional regeneration. Our study reveals that CTGF is necessary and sufficient to stimulate glial bridging and natural spinal cord regeneration.//////////////////
NCBI Summary:
The protein encoded by this gene is a mitogen that is secreted by vascular endothelial cells. The encoded protein plays a role in chondrocyte proliferation and differentiation, cell adhesion in many cell types, and is related to platelet-derived growth factor. Certain polymorphisms in this gene have been linked with a higher incidence of systemic sclerosis. [provided by RefSeq, Nov 2009]
General function
Ligand, Growth factor, Cell adhesion molecule, Cell death/survival, DNA Replication
Comment
Ctgf mRNA is abundantly expressed in granulosa
cells of preantral and early antral follicles (Harlow Hiller 2002 MCE) Additionally,
Ctgf mRNA levels increase in granulosa and
thecal cells of porcine ovaries during early antral follicle
development (Wandji et al 2000). In antral follicles, Ctgf mRNA expression
becomes maximal but is down-regulated in preovulatory
follicles (Harlow Hiller 2002 MCE). FSH reduces Ctgf mRNA expression
in granulosa cells both in vivo and in vitro, yet induces
follicle maturation (Harlow et al 2002 ENdo). In addition, CTGF treatment of
ovaries induces the expression of many genes related to
cell-cycle progression and cell differentiation, thereby enhancing
the growth of immature follicles (Schindler R, Nilsson E, Skinner MK 2010).
Effect of sphingosine-1-phosphate on activation of dormant follicles in murine and human ovarian tissue. Pors SE et al. (2020) In vitro activation of resting ovarian follicles, with the use of mechanical stress and/or pharmacological compounds, is an emerging and novel approach for infertility treatment. The aim of this study was to assess the sphingolipid, sphingosine-1-phosphate (S1P), as a potential in vitro activation agent in murine and human ovarian tissues and isolated follicles. Juvenile murine ovaries and donated human ovarian tissues, from 10 women undergoing ovarian tissue cryopreservation for fertility preservation, were incubated with or without 12 μM S1P for 3 hours for quantitative PCR analysis, and 12 hours for xenotransplantation or culture studies. Gene expression analyses were performed for genes downstream of the Hippo signaling pathway. Murine ovaries and isolated murine and human preantral follicles showed significantly increased mRNA expression levels of Ccn2/CCN2 following S1P treatment compared to controls. This increase was shown to be specific for the Hippo signaling pathway and for the S1P2 receptor, as co-treatment with Hippo-inhibitor, verteporfin, and S1PR2 antagonist, JTE-013, reduced the S1P-induced Ccn2 gene expression in murine ovaries. Histological evaluation of human cortical tissues (5x5x1 mm; n = 30; 3 pieces per patient) xenografted for 6 weeks and juvenile murine ovaries cultured for 4 days (n = 9) or allografted for 2 weeks (n = 48) showed no differences in the distribution of resting or growing follicles in S1P-treated ovarian tissues compared to controls. Collectively, S1P increased Ccn2/CCN2 gene expression in isolated preantral follicles and ovarian tissue from mice and human, but it did not promote follicle activation or growth in vivo. Thus, S1P does not appear to be a potent in vitro activation agent under these experimental conditions.////////////////// CCN2 Mediates S1P-Induced Upregulation of COX2 Expression in Human Granulosa-Lutein Cells. Hu LL et al. (2019) CCN1 and CCN2 are members of the CCN family and play essential roles in the regulation of multiple female reproductive functions, including ovulation. Cyclooxygenase-2 (COX2) is a critical mediator of ovulation and can be induced by sphingosine-1-phosphate (S1P) through the S1P1/3-mediated Yes-associated protein (YAP) signaling. However, it is unclear whether CCN1 or CCN2 can mediate S1P-induced upregulation of COX2 expression and increase in prostaglandin E2 (PGE2) production in human granulosa-lutein (hGL) cells. In the present study, we investigated the effects of S1P on the expressions of CCN1 and CCN2 in hGL cells. Additionally, we used a dual inhibition approach (siRNA-mediated silencing and small molecular inhibitors) to investigate the molecular mechanisms of S1P effects. Our results showed that S1P treatment significantly upregulated the expression of CCN1 and CCN2 in a concentration-dependent manner in hGL cells. Additionally, inhibition or silencing of S1P1, but not S1P3, completely abolished the S1P-induced upregulation of CCN2 expression. Furthermore, we demonstrated that S1P-induced nuclear translocation of YAP and inhibition or silencing of YAP completely abolished the S1P-induced upregulation of CCN1 and CCN2 expression. Notably, silencing of CCN2, but not CCN1, completely reversed the S1P-induced upregulation of COX2 expression and the increase in PGE2 production. Thus, CCN2 mediates the S1P-induced upregulation of COX2 expression through the S1P1-mediated signaling pathway in hGL cells. Our findings expand our understanding of the molecular mechanism underlying the S1P-mediated cellular activities in the human ovary.//////////////////
Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment. Kawamura K 2013 et al.
Primary ovarian insufficiency (POI) and polycystic ovarian syndrome are ovarian diseases causing infertility. Although there is no effective treatment for POI, therapies for polycystic ovarian syndrome include ovarian wedge resection or laser drilling to induce follicle growth. Underlying mechanisms for these disruptive procedures are unclear. Here, we explored the role of the conserved Hippo signaling pathway that serves to maintain optimal size across organs and species. We found that fragmentation of murine ovaries promoted actin polymerization and disrupted ovarian Hippo signaling, leading to increased expression of downstream growth factors, promotion of follicle growth, and the generation of mature oocytes. In addition to elucidating mechanisms underlying follicle growth elicited by ovarian damage, we further demonstrated additive follicle growth when ovarian fragmentation was combined with Akt stimulator treatments. We then extended results to treatment of infertility in POI patients via disruption of Hippo signaling by fragmenting ovaries followed by Akt stimulator treatment and autografting. We successfully promoted follicle growth, retrieved mature oocytes, and performed in vitro fertilization. Following embryo transfer, a healthy baby was delivered. The ovarian fragmentation-in vitro activation approach is not only valuable for treating infertility of POI patients but could also be useful for middle-aged infertile women, cancer patients undergoing sterilizing treatments, and other conditions of diminished ovarian reserve.
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Connective tissue growth factor in the ovarian paracrine system. Harlow CR et al. The endocrine actions of follicle stimulating hormone and luteinising hormone on ovarian cells are transduced by locally produced paracrine factors that regulate the formation of extracellular matrix, proteolytic enzymes and protease inhibitors, which continuously remodel the parenchymal environment in which follicles develop. We recently identified connective tissue growth factor (CTGF) as a gene expressed during the predifferentiated stage of granulosa cell development in rat ovary. The CTGF gene encodes a protein that is implicated in the regulation of connective tissue synthesis, mototaxis, angiogenesis and cellular interaction with ECM at various sites in the body. Stimulation of granulosa cells by FSH in vitro and in vivo induces follicular maturation associated with down-regulation of granulosa cell CTGF mRNA expression. The gene remains expressed in cells of the innermost (antrally located) granulosa compartment up to and after the point of ovulation. Based on the inferred biological properties of CTGF protein and the spatiotemporal pattern of CTGF mRNA expression in the ovary, we postulate roles for ovarian CTGF during early stages of follicular development and after ovulation in the formation of the corpus luteum.
Induction of Ovarian Primordial Follicle Assembly by Connective Tissue Growth Factor CTGF. Schindler R et al. Primordial follicle assembly is a process that occurs when oocyte nests break down to form individual primordial follicles. The size of this initial pool of primordial follicles in part determines the reproductive lifespan of the female. Connective tissue growth factor (CTGF) was identified as a potential regulatory candidate for this process in a previous microarray analysis of follicle development. The current study examines the effects of CTGF and associated transforming growth factor beta 1 (TGFb1) on follicle assembly. Ovaries were removed from newborn rat pups and placed in an organ culture system. The ovaries treated with CTGF for two days were found to have an increased proportion of assembled follicles. CTGF was found to regulate the ovarian transcriptome during primordial follicle assembly and an integrative network of genes was identified. TGFb1 had no effect on primordial follicle assembly and in combination with CTGF decreased oocyte number in the ovary after two days of culture. Over ten days of treatment only the combined treatment of CTGF and TGFb1 was found to cause an increase in the proportion of assembled follicles. Interestingly, treatment with TGF?1 alone resulted in fewer total oocytes in the ovary and decreased the primordial follicle pool size after ten days of culture. Observations indicate that CTGF alone or in combination with TGFb1 stimulates primordial follicle assembly and TGF?1 can decrease the primordial follicle pool size. These observations suggest the possibility of manipulating primordial follicle pool size and influencing female reproductive lifespan.
Christopher R. Harlow, et al 2002 reported that FSH and TGF-beta Superfamily Members Regulate Granulosa Cell Connective Tissue Growth Factor Gene Expression in Vitro and in Vivo.
:
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. 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 FSH?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 ?M 5 -dihydrotestosterone), consistent with mediation by intracellular cAMP. Conversely, treatment of granulosa cell cultures with TGF? (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 TGF? (10 ng/ml) on CTGF mRNA, but substantially enhanced aromatase activity beyond levels induced by FSH alone. Importantly, other TGF?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 TGF? (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 TGF?, growth/differentiation factor-9, and activin, suggesting paracrine roles for these growth/differentiation factors in the regulation of CTGF synthesis in mammalian ovaries.
.........
Connective tissue growth factor (CTGF) expression in the human corpus luteum: paracrine regulation by human chorionic gonadotropin (hCG) Duncan WC, et al .
Context: The molecular mechanisms of luteolysis and its inhibition during maternal recognition of pregnancy remain unclear. Objective: To investigate the differential regulation of connective tissue growth factor (CTGF) expression in human corpora lutea using in vivo and in vitro models. Design: Corpora lutea from different stages of the luteal phase and after luteal rescue with human chorionic gonadotropin (hCG). Primary cultures and co-cultures of luteinised granulosa cells and luteal fibroblast-like cells. Setting: Research Center of university teaching hospital. Patients: Women with regular cycles having hysterectomy for non-malignant conditions. Women undergoing oocyte collection for assisted conception. Interventions: CTGF localization by in situ hybridization and expression by quantitative RT-PCR. Outcomes: Effect of hCG on the expression and localization of CTGF mRNA in human corpora lutea. Effect of hCG on CTGF expression in primary cultures of luteinised granulosa cells and luteal fibroblast-like cells. Results: Luteal rescue reduced CTGF expression when compared with the late-luteal phase (P < 0.05). CTGF expression was localized to fibroblast-like cells and endothelial cells of larger blood vessels and not to steroidogenic cells. The expression of CTGF by fibroblast-like cells in vitro is not regulated by hCG. When co-cultured with luteinized granulosa cells, fibroblast-like cell CTGF expression was inhibited by hCG (P < 0.001). This effect is independent of stimulated progesterone concentrations and is not blocked by follistatin or indomethacin. Both interleukin-1alpha (P < 0.05) and cyclic AMP (P < 0.001), inhibited CTGF expression in fibroblast-like cells. Conclusions: These results provide evidence for negative regulation of CTGF by hCG during luteal rescue mediated by paracrine signals.
.....................
Gene bionetwork analysis of ovarian primordial follicle development. Nilsson EE et al. Ovarian primordial follicles are critical for female reproduction and comprise a finite pool of gametes arrested in development. A systems biology approach was used to identify regulatory gene networks essential for primordial follicle development. Transcriptional responses to eight different growth factors known to influence primordial follicles were used to construct a bionetwork of regulatory genes involved in rat primordial follicle development. Over 1,500 genes were found to be regulated by the various growth factors and a network analysis identified critical gene modules involved in a number of signaling pathways and cellular processes. A set of 55 genes was identified as potential critical regulators of these gene modules, and a sub-network associated with development was determined. Within the network two previously identified regulatory genes were confirmed (i.e., Pdgfa and Fgfr2) and a new factor was identified, connective tissue growth factor (CTGF). CTGF was tested in ovarian organ cultures and found to stimulate primordial follicle development. Therefore, the relevant gene network associated with primordial follicle development was validated and the critical genes and pathways involved in this process were identified. This is one of the first applications of network analysis to a normal developmental process. These observations provide insights into potential therapeutic targets for preventing ovarian disease and promoting female reproduction.
decreases after preovualtory hCG in mice. (array data)
/////
BMP6 increases CD68 expression by up-regulating CTGF expression in human granulosa-lutein cells. Zhang XY et al. (2021) Bone morphogenetic protein 6 (BMP6) and connective tissue growth factor (CTGF) are critical growth factors required for normal follicular development and luteal function. Cluster of Differentiation 68 (CD68) is an intraovarian marker of macrophages that plays an important role in modulating the physiological regression of the corpus luteum. The aim of this study was to investigate the effect of BMP6 on the expression of CTGF and the subsequent increase in CD68 expression as well as its underlying mechanisms. Primary and immortalized (SVOG) human granulosa cells obtained from infertile women undergoing in vitro fertilization treatment were used as cell models to conduct the in vitro experiments. Our results showed that BMP6 treatment significantly increased the expression levels of CTGF and CD68. Using BMP type I receptor inhibitors (dorsomorphin, DMH-1 and SB431542), we demonstrated that both activin receptor-like kinase (ALK)2 and ALK3 are involved in BMP6-induced stimulatory effects on the expression of CTGF and CD68. Additionally, SMAD4-knock down reversed the BMP6-induced up-regulation of CTGF and CD68, indicating that the canonical SMAD signaling pathway is required for these effects. Moreover, CTGF-knock down abolished the BMP6-induced up-regulation of CD68 expression. These findings indicate that intrafollicular CTGF mediates BMP6-induced increases in CD68 expression through the ALK2/ALK3-mediated SMAD-dependent signaling pathway./////////////////////SMAD-dependent signaling mediates BMP6-induced stimulation of connective tissue growth factor in luteinized human granulosa cells. Liu S et al. (2019) Connective tissue growth factor (also known as CTGF or CCN2) is a secreted matricellular protein that belongs to the CCN family. With wide-ranging biological activities and tissue expression patterns, CTGF plays a critical role in regulating various cellular functions. In the female reproductive system, CTGF is highly expressed in granulosa cells in growing ovarian follicles and is involved in the regulation of follicular development, ovulation, and luteal function. In the mammalian ovary, bone morphogenetic protein 6 (BMP6) is an important intraovarian modulator of follicular development. In this study, we demonstrated that BMP6 treatment significantly increased the expression of CTGF in both primary and immortalized human granulosa cells. Using both pharmacological inhibitors and siRNA-mediated knockdown approaches, we showed that ALK2 and ALK3 type I receptors are required for BMP6-induced cellular activities. Furthermore, this effect is most likely mediated by a SMAD-dependent pathway. Our studies provide novel insight into the molecular mechanisms by which an intraovarian growth factor affects the production of another factor via a paracrine effect in human granulosa cells.//////////////////Connective tissue growth factor mediates growth differentiation factor 8-induced increase of lysyl oxidase activity in human granulosa-lutein cells. Chang HM et al. (2016) Lysyl oxidase (LOX) is an essential enzyme for the stabilization of the extracellular matrix (ECM) and the subsequent follicle and oocyte maturation. Currently, there is limited information pertaining to the regulation of LOX activity in human ovarian tissue. Growth differentiation factor 8 (GDF8) is a unique member of the transforming growth factor-β superfamily that is expressed in human granulosa cells and has important roles in regulating a variety of ovarian functions. The aim of the present study was to investigate the effects of GDF8 on the regulation of LOX expression and activity in human granulosa cells and to examine the underlying molecular determinants. An established immortalized human granulosa cell line (SVOG) and primary granulosa-lutein cells were used as study models. Using dual inhibition approaches (TGF-β type I inhibitor SB505124 and small interfering RNAs) and ChIP analyses, we have demonstrated that GDF8 up-regulated the expression of connective tissue growth factor (CTGF) through the activin receptor-like kinase 5-mediated SMAD2/3-SMAD4 signaling pathways. In addition, the increase in CTGF expression contributed to the GDF8-induced increase in LOX expression and activity. Our findings suggest that GDF8 and CTGF may play critical roles in the regulation of ECM formation in human granulosa cells.//////////////////Growth differentiation factor 8 suppresses cell proliferation by up-regulating CTGF expression in human granulosa cells. Chang HM et al. (2015) Connective tissue growth factor (CTGF) is a matricellular protein that plays a critical role in the development of ovarian follicles. Growth differentiation factor 8 (GDF8) is mainly, but not exclusively, expressed in the mammalian musculoskeletal system and is a potent negative regulator of skeletal muscle growth. The aim of this study was to investigate the effects of GDF8 and CTGF on the regulation of cell proliferation in human granulosa cells and to examine its underlying molecular determinants. Using dual inhibition approaches (inhibitors and small interfering RNAs), we have demonstrated that GDF8 induces the up-regulation of CTGF expression through the activin receptor-like kinase (ALK)4/5-mediated SMAD2/3-dependent signaling pathways. In addition, the increase in CTGF expression contributes to the GDF8-induced suppressive effect on granulosa cell proliferation. Our findings suggest that GDF8 and CTGF may play critical roles in the regulation of proliferative events in human granulosa cells.//////////////////
TGF-β1 Up-Regulates Connective Tissue Growth Factor Expression in Human Granulosa Cells through Smad and ERK1/2 Signaling Pathways. Cheng JC et al. (2015) Connective tissue growth factor (CTGF), which is also called CCN2, is a secreted matricellular protein. CTGF regulates various important cellular functions by interacting with multiple molecules in the microenvironment. In the ovary, CTGF is mainly expressed in granulosa cells and involved in the regulation of follicular development, ovulation and luteinization. TGF-β1 has been shown to up-regulate CTGF expression in rat and hen granulosa cells. However, the underlying molecular mechanisms of this up-regulation remain undefined. More importantly, whether the stimulatory effect of TGF-β1 on CTGF expression can be observed in human granulosa cells remains unknown. In the present study, our results demonstrated that TGF-β1 treatment up-regulates CTGF expression in both immortalized human granulosa cells and primary human granulosa cells. Using a siRNA-mediated knockdown approach and a pharmacological inhibitor, we demonstrated that the inhibition of Smad2, Smad3 or ERK1/2 attenuates the TGF-β1-induced up-regulation of CTGF. This study provides important insights into the molecular mechanisms that mediate TGF-β1-up-regulated CTGF expression in human granulosa cells.//////////////////
Differential expression of CTGF in pre- and post-ovulatory granulosa cells in the hen ovary is regulated by TGF? and gonadotrophins. Zhu G et al. Connective tissue growth factor (CTGF) is a cysteine-rich, matrix-associated heparin-binding protein that is important in many cell types as a regulator of cell proliferation, angiogenesis, cell remodelling and other cellular processes. CTGF is necessary for normal follicle growth and luteinisation in mammals. The avian follicular hierarchy provides an excellent experimental model to study developmental events, particularly the role of cellular remodelling factors in the process of folliculogenesis. In this study, we examined CTGF expression and regulation in the hen ovary. CTGF expression was increased considerably as follicular development proceeds in pre-ovulatory follicles, peaking in expression at the time of ovulation. Immunohistochemistry revealed that CTGF protein was concentrated in the cytoplasm of follicular granulosa cells throughout the ovulation cycle. We isolated granulosa cells from the follicles at two key stages of the ovulation cycle (in terms of cellular alteration): during pre-ovulatory growth and during post-ovulatory regression. Follicle-stimulating hormone (FSH) and luteinising hormone (LH) inhibited CTGF expression in pre-ovulatory granulosa cells but stimulated CTGF expression in post-ovulatory granulosa cells. Moreover, TGF? stimulated CTGF expression in both pre- and post-ovulatory granulosa cells. Nevertheless, TGF? could rescue the inhibition of gonadotrophins on pre-ovulatory granulosa CTGF expression but could not further stimulate CTGF expression in gonadotrophin-treated post-ovulatory granulosa cells. The results of this study indicate that CTGF expression in avian granulosa cells is modulated by a combination of gonadotrophins and TGF? according to the different stages of follicle maturation and degradation. The results also suggest that the gonadotrophic action on post-ovulatory follicles in the avian ovary differs from the gonadotrophin-induced luteinisation in mammals.
Liu J, et al 2002 reported that gonadotrophins inhibit the expression of insulin-like growth factor
binding protein-related protein-2 mRNA in cultured human
granulosa-luteal cells.
Insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP) have been shown
to be involved in ovarian follicular growth/development and steroidogenesis.
Recently, a number of low-affinity IGFBP-related proteins (IGFBP-rP) have been
characterized. In this study, the authors investigated the expression of the gene for IGFBP-rP2
(also known as connective tissue growth factor, CTGF) in human granulosa cells in
vitro and in vivo. Northern blot analysis demonstrated that IGFBP-rP2 mRNA is
expressed in cultured human granulosa-luteal cells obtained from women undergoing
an IVF programme. Accumulation of IGFBP-rP2 mRNA was dose-dependently
down-regulated by FSH and LH after 24 h treatment (both P < 0.05) in cultured
granulosa-luteal cells. The inhibitory effects of gonadotrophins were mimicked by
treatment with the protein kinase A activator, (Bu)(2)cAMP. Protein kinase C
inhibitor staurosporine reduced, whereas protein kinase C activator TPA
(12-O-tetradecanoyl phorbol 13-acetate) increased, IGFBP-rP2 mRNA accumulation.
These results suggest that the inhibitory effects of gonadotrophins on IGFBP-rP2 gene
expression may involve signal transduction via both protein kinase A and C pathways.
Immunohistochemical analysis revealed positive staining for IGFBP-rP2 in the
granulosa and theca cells of normal human ovarian follicles. Corpus luteum and
ovarian surface epithelial cells were also positively stained. Modulation of
IGFBP-rP2 expression by gonadotrophic hormones may have a role in ovarian
follicular development and in the ovulatory process.
Hillier ET AL 2001 studied the regulation of Connective Tissue Growth Factor and
Lysyl Oxidase Gene Expression in Rat granulosa Cells.
They
used Differential Display RT-PCR (DDRT-PCR) to identify novel
FSH/androgen-regulated genes in cultured rat granulosa cells (GC).
23-Day old female Wistar rats received diethylstilbestrol
injections for 2d to stimulate preantral follicular
development. Isolated GC were cultured in serum free medium
containing no hormone (control), FSH (10ng ml-1),
5alpha-dihydrotestosterone (DHT) (10-6 M) or FSH+DHT. Cells
were harvested at 0, 3, 12, 24 and 48h of incubation and total RNA
extracted. The DDRT-PCR screen generated six distinct cDNA clones that reproduce the
DDRT-PCR profile on a Northern blot of the corresponding RNA
samples. Two of these clones detect transcripts that are strongly
down-regulated by FSH alone and further suppressed in the
presence of FSH/androgen. One corresponds to connective tissue
growth factor (CTGF) a secreted protein implicated in tissue
remodelling and angiogenesis, and a second is identical to lysyl
oxidase (LO), a key participant in extracellular matrix deposition.
In detailed time-course studies, Northern analysis reveals a single
~2.5kb CTGF transcript that is maximally suppressed within 3h
exposure to FSH+DHT and two LO transcripts (~3.8 and ~5.2kb)
that are maximally suppressed at 48h. An index mRNA transcript
(cytochrome P450arom/CYP19) is maximally induced by FSH+DHT
at 48h in this culture system. In vivo, CTGF and LO transcripts are
also markedly reduced in GC RNA 48h after eCG injection (10 IU
i.p.) compared with untreated controls, and further reduced 12h
after additional 10 IU hCG to induce luteinisation. These results
identify CTGF and LO as GC genes that are negatively regulated by
gonadotropins and androgen.
hCG-dependent regulation of angiogenic factors in human granulosa lutein cells. Phan B et al. As prerequisite for development and maintenance of many diseases angiogenesis is of particular interest in medicine. Pathologic angiogenesis takes place in chronic arthritis, collagen diseases, arteriosclerosis, retinopathy associated with diabetes, and particularly in cancers. However, angiogenesis as a physiological process regularly occurs in the ovary. After ovulation the corpus luteum is formed by rapid vascularization of initially avascular granulosa lutein cell tissue. This process is regulated by gonadotropic hormones. In order to gain further insights in the regulatory mechanisms of angiogenesis in the ovary, we investigated these mechanisms in cell culture of human granulosa lutein cells. In particular, we determined the expression and production of several angiogenic factors including tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), Leptin, connective tissue growth factor (CTGF), meningioma-associated complimentary DNA (Mac25), basic fibroblast growth factor (bFGF), and Midkine. In addition, we showed that human chorionic gonadotropin (hCG) has distinct effects on their expression and production. hCG enhances the expression and production of TIMP-1, whereas it downregulates the expression of CTGF and Mac25. Furthermore it decreases the expression of Leptin. Our results provide evidence that hCG determines growth and development of the corpus luteum by mediating angiogenic pathways in human granulosa lutein cells. Hence we describe a further approach to understand the regulation of angiogenesis in the ovary. Mol. Reprod. Dev. (c) 2006 Wiley-Liss, Inc.
Ovarian localization
Granulosa, Theca, Luteal cells
Comment
S.-A. Wandji, et al 2000 reported that messenger RNAs for MAC25 and Connective Tissue Growth Factor
(CTGF) Are Inversely Regulated during Folliculogenesis and Early Luteogenesis.
CTGF transcripts were induced during the late preantral stage in granulosa and theca cells
concomitantly with the appearance of endothelial cells in the theca. CTGF mRNA expression increased in granulosa cells to
a maximum in mid-antral follicles but was down regulated in preovulatory follicles. In contrast,
granulosa cell mac25 mRNA expression was undetectable between the preantral and mid-antral stage but was strongly
induced in terminally differentiated granulosa cells of preovulatory follicles. CTGF mRNA and peptide were also detected
in the theca externa/interstitium and in vascular endothelial cells of ovarian blood vessels, whereas mac25 transcripts, which
were also abundant in ovarian blood vessels increased in the theca interna with follicular development. There was a marked
up-regulation of CTGF mRNA expression in granulosa luteins concomitantly with an increase in endothelial cell
proliferation within the CL. IT was hypothesize that CTGF may promote ovarian cell growth and blood vessel formation during
follicular and luteal development whereas mac25, a tumor inhibitor, may promote terminal differentiation of granulosa cells
in preovulatory follicles.
Connective tissue growth factor (CTGF) expression in the human corpus luteum: paracrine regulation by human chorionic gonadotropin (hCG) Duncan WC, et al .
Context: The molecular mechanisms of luteolysis and its inhibition during maternal recognition of pregnancy remain unclear. Objective: To investigate the differential regulation of connective tissue growth factor (CTGF) expression in human corpora lutea using in vivo and in vitro models. Design: Corpora lutea from different stages of the luteal phase and after luteal rescue with human chorionic gonadotropin (hCG). Primary cultures and co-cultures of luteinised granulosa cells and luteal fibroblast-like cells. Setting: Research Center of university teaching hospital. Patients: Women with regular cycles having hysterectomy for non-malignant conditions. Women undergoing oocyte collection for assisted conception. Interventions: CTGF localization by in situ hybridization and expression by quantitative RT-PCR. Outcomes: Effect of hCG on the expression and localization of CTGF mRNA in human corpora lutea. Effect of hCG on CTGF expression in primary cultures of luteinised granulosa cells and luteal fibroblast-like cells. Results: Luteal rescue reduced CTGF expression when compared with the late-luteal phase (P < 0.05). CTGF expression was localized to fibroblast-like cells and endothelial cells of larger blood vessels and not to steroidogenic cells. The expression of CTGF by fibroblast-like cells in vitro is not regulated by hCG. When co-cultured with luteinized granulosa cells, fibroblast-like cell CTGF expression was inhibited by hCG (P < 0.001). This effect is independent of stimulated progesterone concentrations and is not blocked by follistatin or indomethacin. Both interleukin-1alpha (P < 0.05) and cyclic AMP (P < 0.001), inhibited CTGF expression in fibroblast-like cells. Conclusions: These results provide evidence for negative regulation of CTGF by hCG during luteal rescue mediated by paracrine signals.
Follicle stages
Antral, Preovulatory
Comment
Oestrogen formation and connective tissue growth factor expression in rat granulosa cells. Harlow CR et al. Ovarian follicular development involves continual remodelling of the extracellular matrix (ECM) forming the basement membrane and intercellular framework that support granulosa cell (GC) growth and differentiation. Insight into the molecular regulation of ovarian ECM remodelling is potentially translatable to tissue remodelling elsewhere in the body. We therefore studied the link between a gene marker of ECM remodelling (connective tissue growth factor (CTGF)) and oestrogen biosynthesis (cytochrome P450(aromatase) (P450(arom))) in rat granulosa cells. To determine if a cause-effect interaction exists, we used semi-quantitative in situ hybridisation to analyse patterns of CTGF and P450(arom) mRNA expression and immunohistochemistry to detect CTGF protein localisation throughout follicular development, and tested the actions of CTGF on oestrogen biosynthesis and oestradiol on CTGF mRNA expression in isolated GC in vitro. CTGF mRNA levels in GC rose gradually through small preantral (SP) and small antral (SA) stages of development to a maximum (fivefold higher) in large antral (LA) follicles. In preovulatory (PO) follicles, the CTGF mRNA level fell to 30% of that in SP follicles. P450(arom) mRNA also increased (threefold in LA relative to SP) throughout antral development follicles, but in contrast to CTGF continued to increase (12-fold) in PO follicles. In the cumulus oophorus of PO follicles, the residual GC CTGF mRNA expression increased with proximity to the oocyte, being inversely related to P450(arom). Elsewhere in the follicle wall, there was a mural-to-antral gradient of CTGF mRNA expression, again inversely related to P450(arom). Immunohistochemistry showed CTGF protein localisation broadly followed mRNA expression during follicular development, although the protein was also present in the theca interna and ovarian surface epithelium. Gradients in CTGF expression across the cumulus oophorus and follicle wall were similar to those observed for mRNA with CTGF protein expression being greatest in proximity to the oocyte. Treatment of isolated GC from preantral and SA follicles with recombinant human CTGF (1-100 ng/ml) did not affect basal or FSH-stimulated GC aromatase activity. However, in the absence of FSH, oestradiol (10(-7)-10(-5) M) stimulated CTGF mRNA expression up to twofold. Conversely, FSH (10 ng/ml) alone reduced CTGF mRNA expression by 40% and combined treatment with FSH and oestradiol further suppressed CTGF to 10% of the control value. The oestrogen receptor (ER) antagonist, ICI 182 780 blocked the stimulatory and inhibitory effects of oestradiol, suggesting a specific ER-mediated mode of action on CTGF. Therefore, CTGF gene expression in GC is under local control by oestrogen whose effect (positive or negative) is modulated by FSH. This helps explain why gene expression of CTGF and P450(arom) diverge in FSH-induced PO follicles and has implications for oestrogenic regulation of CTGF formation elsewhere in the body.
Phenotypes
Mutations
2 mutations
Species: mouse
Mutation name: None
type: null mutation fertility: subfertile Comment: Connective Tissue Growth Factor Is Required for Normal Follicle Development and Ovulation. Nagashima T et al. Connective tissue growth factor (CTGF) is a cysteine-rich protein the synthesis and secretion of which are hypothesized to be selectively regulated by activins and other members of the TGF-?superfamily. To investigate the in vivo roles of CTGF in female reproduction, we generated Ctgf ovarian and uterine conditional knockout (cKO) mice. Ctgf cKO mice exhibit severe subfertility and multiple reproductive defects including disrupted follicle development, decreased ovulation rates, increased numbers of corpus luteum, and smaller but functionally normal uterine horns. Steroidogenesis is disrupted in the Ctgf cKO mice, leading to increased levels of serum progesterone. We show that disrupted follicle development is accompanied by a significant increase in granulosa cell apoptosis. Moreover, despite normal cumulus expansion, Ctgf cKO mice exhibit a significant decrease in oocytes ovulated, likely due to impaired ovulatory process. During analyses of mRNA expression, we discovered that Ctgf cKO granulosa cells show gene expression changes similar to our previously reported granulosa cell-specific knockouts of activin and Smad4, the common TGF-bfamily intracellular signaling protein. We also discovered a significant down-regulation of Adamts1, a progesterone-regulated gene that is critical for the remodeling of extracellular matrix surrounding granulosa cells of preovulatory follicles. These findings demonstrate that CTGF is a downstream mediator in TGF-?and progesterone signaling cascades and is necessary for normal follicle development and ovulation....................As shown in Fig.
3G, the follicle count showed reduced numbers of preantral
and antral follicles , and a trend toward increased numbers of atretic
preantral and
atretic antral follicles in Ctgf cKO (Amhr2cre/) ovaries
compared with control ovaries. There were also significant increase of granulosa cell
apoptosis in Ctgf cKO female mice.
Species: human
Mutation name: type: naturally occurring fertility: subfertile Comment: Identification of the key regulating genes of diminished ovarian reserve (DOR) by network and gene ontology analysis. Pashaiasl M et al. (2016) Diminished ovarian reserve (DOR) is one of the reasons for infertility that not only affects both older and young women. Ovarian reserve assessment can be used as a new prognostic tool for infertility treatment decision making. Here, up- and down-regulated gene expression profiles of granulosa cells were analysed to generate a putative interaction map of the involved genes. In addition, gene ontology (GO) analysis was used to get insight intol the biological processes and molecular functions of involved proteins in DOR. Eleven up-regulated genes and nine down-regulated genes were identified and assessed by constructing interaction networks based on their biological processes. PTGS2, CTGF, LHCGR, CITED, SOCS2, STAR and FSTL3 were the key nodes in the up-regulated networks, while the IGF2, AMH, GREM, and FOXC1 proteins were key in the down-regulated networks. MIRN101-1, MIRN153-1 and MIRN194-1 inhibited the expression of SOCS2, while CSH1 and BMP2 positively regulated IGF1 and IGF2. Ossification, ovarian follicle development, vasculogenesis, sequence-specific DNA binding transcription factor activity, and golgi apparatus are the major differential groups between up-regulated and down-regulated genes in DOR. Meta-analysis of publicly available transcriptomic data highlighted the high coexpression of CTGF, connective tissue growth factor, with the other key regulators of DOR. CTGF is involved in organ senescence and focal adhesion pathway according to GO analysis. These findings provide a comprehensive system biology based insight into the aetiology of DOR through network and gene ontology analyses.//////////////////