Molecular Human Reproduction, Vol. 8, No. 2, 136-141,
February 2002
© 2002 European Society of Human Reproduction and Embryology
Ovary and oogenesis |
Gonadotrophins inhibit the expression of insulin-like growth factor binding protein-related protein-2 mRNA in cultured human granulosa–luteal cells
1 Department of Pathology, P.O.Box 21, University of Helsinki, FIN-00014 Helsinki, 2 Department of Paediatrics and 3 Department of Pathology and Forensic Medicine, P.O.Box 1777, Kuopio University Hospital and University of Kuopio, FIN-70211 Kuopio and 4 Department of Obstetrics and Gynaecology, P.O.Box 140, Helsinki University Central Hospital, FIN-00290 Helsinki, Finland
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Abstract |
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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, we 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)2cAMP. 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.
gonadotrophin/granulosa-luteal/IGFBP-rP/ovary/protein kinase
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Insulin-like growth factors (IGF) are involved in ovarian follicular development and steroidogenesis (Adashi et al., 1991
; Giudice, 1992; Wang and Chard, 1999). Human granulosa cells express the IGF-II gene, which is regulated by gonadotrophins and cAMP analogues (Voutilainen and Miller, 1987), and implicated in granulosa cell proliferation (Di Blasio et al., 1994) and progesterone production (Kamada et al., 1992; Devoto et al., 1999). IGF are bound to high-affinity IGF-binding proteins (IGFBP) in serum and other biological fluids. To date, six high-affinity binding proteins (IGFBP-1 to IGFBP-6) have been identified and characterized (Hwa et al., 1999). The physiological roles of IGFBP include prevention of the hypoglycaemic effects of IGF, prolongation of IGF clearance time, transportation of IGF to appropriate sites of action, and modulation of the actions of IGF on cells (Hwa et al., 1999). These classical IGFBP are expressed in granulosa cells, and gonadotrophins may regulate their expression (Voutilainen et al., 1996; Wang and Chard, 1999).
In addition to the six-high affinity IGFBP, a number of other proteins bearing structural similarities with the IGFBP can bind IGF, but with a substantially lower affinity than the classical IGFBP. The N-terminal domains of these low-affinity proteins are homologous to the classical IGFBP, and this region has long been considered to be critical for IGF binding. These findings have led to the proposal of low-affinity IGFBP-related proteins (IGFBP-rP), including IGFBP-rP1 (also known as mac25) and IGFBP-rP2 (also known as connective tissue growth factor, CTGF) (Hwa et al., 1999). The IGFBP-rP2 gene is expressed in porcine and rat granulosa cells, and its transcripts are suppressed by exposure to FSH and chorionic gonadotrophin (Wandji et al., 2000; Slee et al., 2001). IGFBP-rP2 protein has also been detected in human follicular fluid (Yang et al., 1998). Due to the importance of the IGF system in ovarian physiology, we were particularly interested in looking at the possible expression and regulation of these new members of the IGFBP superfamily in human ovaries. We show here that the IGFBP-rP2 gene is expressed in human granulosa–luteal cells, and that its expression is inhibited by gonadotrophin treatment. Signal transduction through both protein kinase A and C pathways seems to be involved in the regulation of IGFBP-rP2 gene expression.
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Materials and methods |
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Cell cultures
Human granulosa–luteal cells were harvested during follicular aspiration from women undergoing oocyte retrieval for IVF, as described previously (Liu et al., 2001
). The study protocol was approved by the Research Ethics Committees of Helsinki and Kuopio University Hospitals, and the women gave informed written consent. The cells obtained from three to eight patients in the same morning were pooled, enzymatically dispersed, separated from red blood cells, and cultured in 1:1 Dulbecco's modified Eagle's medium (DMEM)–Ham's F-12 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal calf serum (FCS; Gibco), 2 mmol/l L-glutamine, and antibiotics (100 IU/ml penicillin and 100 µg/ml streptomycin) at 37°C in a 95% air–5% CO2 humidified environment. Cell culture media were replaced every other day. Recombinant human FSH (rhFSH) and LH (rhLH) were gifts from Serono-Nordic (Vantaa, Finland), and recombinant human activin A peptide was generously provided by Dr A.F.Parlow, NIDDK National Hormone and Pituitary Program (USA). (Bu)2cAMP and 12-O-tetradecanoyl phorbol 13-acetate (TPA) were purchased from Sigma Chemical Co. (St Louis, MO, USA), and staurosporine was from Boehringer Mannheim (Mannheim, Germany).
RNA analysis
Extraction of cytoplasmic RNA, Northern blotting, and hybridization conditions were the same as previously described (Liu et al., 1996). A 30 mer oligonucleotide probe was used to detect the IGFBP-rP2 mRNA by Northern hybridization. The oligonucleotide sequence for IGFBP-rP2 was 5'-CTTCATGCCATGTCTCCGTACATCTTCCTG-3', complementary to nucleotides 1168–1197 of the human CTGF mRNA (GenBank accession no. NM_001901) (Oemar et al., 1997). Ribosomal 28S RNA cDNA was used for controlling RNA loading (Arnheim, 1979). The oligonucleotide and cDNA probes were labelled as described previously (Liu et al., 1996). The relative intensities of autoradiographic signals were quantified by densitometric scanning. All mRNA data shown were normalized with the respective 28S RNA values and repeated at least three times on different batches of cells from different patients.
Immunohistochemistry
The paraffin sections of three normal human ovarian tissues were deparaffinized and rehydrated using xylene and graded alcohols. Antigen retrieval was accomplished by heating in a microwave oven at 800 W in a citrate buffer (pH 6.0) for 3x5 min. After washing in phosphate-buffered saline (pH 7.2), endogenous peroxidase activity was blocked with 5% hydrogen peroxide for 5 min. After normal serum (Vectastain Elite ABC Kit PK-6105 Goat; Vector Laboratories, Burlingame, CA, USA) incubation, non-specific binding was blocked with avidin, followed by biotin (Blocking Kit SP-2001; Vector). The sections were then incubated overnight at 4°C with the primary anti-CTGF antibody (R&D Systems, Abingdon, Oxon, UK; 1:30 dilution). After another washing step, the bound antibody was localized using a biotinylated secondary antibody and an avidin–biotin–peroxidase detection kit (Vectastain Elite ABC Kit PK-6105 Goat; Vector), in which diaminobenzidine tetrahydrochloride (DAB; Sigma) was used as a chromogen. Finally, the samples were slightly counterstained with Mayer's haematoxylin, dehydrated, and mounted with Depex (BDH Laboratory Supplies, Poole, Dorset, UK). In each staining batch a human adrenal gland specimen was used as a positive control, and an ovarian section processed without the primary antibody was used as a negative control.
Progesterone measurement
Progesterone was measured by a competitive enzyme immunoassay (EIA) purchased from Diagnostic Systems Laboratories, Inc. (Webster, TX, USA) according to the manufacturer's instructions. The detection limit of the assay was considered 1 nmol/l. The intra- and inter-assay coefficients of variation were 7.5 and 9.4%, respectively.
Statistics
Differences in the IGFBP-rP2 mRNA levels and progesterone concentrations were assessed by the Mann–Whitney test. The level of significance was chosen as P < 0.05.
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Results |
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We cultured the granulosa–luteal cells for 5–10 days before initiation of hormonal stimulations. At this culture stage, progesterone production is optimally responsive to gonadotrophin treatment (Voutilainen et al., 1986
; Schipper et al., 1993). Basal secretion of progesterone by the cultured cells without any stimulation was in a range of 0.9–9 nmol per day per well (3 ml medium per well). Treatment with gonadotrophins rhFSH (100 IU/l) or rhLH (100 IU/l) for 24 h increased progesterone secretion to 270 ± 40% (mean ± SEM) (P < 0.005, n = 5) and 298 ± 68% (P < 0.01, n = 4 ) of the control, respectively (Figure 1). Incubation with the protein kinase A activator, (Bu)2cAMP (1 mmol/l) stimulated progesterone secretion to 377 ± 83% of the control (P < 0.01, n = 4; Figure 1). The stimulatory effects of gonadotrophins and (Bu)2cAMP on progesterone secretion were detectable already at the 3 h time point, with a time-dependent increase until 48 h of treatment (data not shown).
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Expression of IGFBP-rP2 mRNA was detectable by Northern blotting, and the transcript was ~2.4 kb in size (Figure 2A
), the same size as reported previously (Nakanishi et al., 1997; Oemar et al., 1997). Besides granulosa–luteal cells, IGFBP-rP2 mRNA was also detectable in other human steroidogenic tissues, including testes, a Leydig cell tumour, adrenals, adrenocortical tumours and in the adrenocortical cell line NCI-H295R (data not shown). In cultured granulosa–luteal cells, the IGFBP-rP2 mRNA expression level was not influenced by the serum concentration in the medium (data not shown). The accumulation of IGFBP-rP2 mRNA was reduced (to ~15–40% of the control levels) by rhFSH and rhLH (both at 100 IU/l) after 24 h of treatment (n = 7 and n = 6, respectively; both P < 0.05). These inhibitory effects of LH and FSH on IGFBP-rP2 gene expression were dose-dependent (Figures 2B and 3). The effect of the gonadotrophins was not yet detectable after 2 h of treatment, reached its maximum at 24 h of treatment, and was maintained at the same level until at least the 48 h time point.
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Since the protein kinase A pathway is the main signal transduction system involved in the induction of steroidogenesis by gonadotrophins in human granulosa–luteal cells, we investigated whether the inhibitory effects of LH and FSH on IGFBP-rP2 mRNA accumulation involve the protein kinase A signal transduction pathway. As shown in Figure 2C
, the effect of gonadotrophins on IGFBP-rP2 gene expression was mimicked by (Bu)2cAMP treatment, suggesting that the protein kinase A pathway may be involved in the inhibitory effect of the gonadotrophins. However, the effect of (Bu)2cAMP on IGFBP-rP2 mRNA accumulation was much stronger than that of gonadotrophins, suggesting that there may be other signal transduction pathways activated by cAMP. Therefore, we studied the effects of protein kinase C modulators on IGFBP-rP2 expression. Treatment for 24 h with the protein kinase C inhibitor staurosporine (50 nmol/l) (Figure 2A) blocked totally the expression of IGFBP-rP2 mRNA (n = 3, P < 0.05). The inhibitory effect of staurosporine was dose-dependent, and already detectable at the concentration of 1 nmol/l (data not shown). Conversely, the protein kinase C activator TPA dose-dependently (1.5–160 nmol/l) increased the expression of IGFBP-rP2 mRNA up to 377% of that of the control, with a half maximal increase at ~50 nmol/l (data not shown). Treatment with activin A (100 ng/ml) had no significant effect on IGFBP-rP2 mRNA accumulation (data not shown).
We used immunohistochemistry to analyse IGFBP-rP2 protein expression in three normal human ovaries. IGFBP-rP2 immunoreactivity was seen in the cytoplasm and nuclei of the granulosa cells and partly in the theca cells of ovarian follicles, indicating that IGFBP-rP2 mRNA is efficiently translated (Figure 4). Corpora lutea and ovarian surface epithelial cells were also positive for IGFBP-rP2. As a positive control, human adrenal cortex showed consistent positive immunohistochemical staining (Figure 4).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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In this study, we showed that human ovarian granulosa–luteal cells express the IGFBP-rP2 gene, confirming the previous reports of porcine and rat studies (Wandji et al., 2000
; Slee et al., 2001). Besides granulosa–luteal cells, we detected IGFBP-rP2 mRNA in other human steroidogenic tissues, including the testes and the adrenals. In cultured human granulosa–luteal cells, FSH and LH down-regulated IGFBP-rP2 mRNA accumulation, in contrast with their stimulatory effects on progesterone secretion and steroidogenic enzyme gene expression (Voutilainen et al., 1986; Strauss and Steinkampf, 1995). It is well known that the actions of FSH and LH are mediated principally through the activation of adenylate cyclase and the subsequent increase in intracellular cAMP levels. Consistently, treatment with (Bu)2cAMP efficiently inhibited the expression of the IGFBP-rP2 gene, suggesting that the protein kinase A pathway is involved in the inhibitory effect of gonadotrophins on IGFBP-rP2 gene expression in cultured granulosa–luteal cells. This is in agreement with previous reports demonstrating that transforming growth factor-ß (TGF-ß) induced expression of IGFBP-rP2 in human foreskin fibroblast cultures was almost completely blocked by 8-Br-cAMP (Kothapalli et al., 1998; Duncan et al., 1999). In addition, the response to both protein kinase C inhibitor staurosporine, and activator TPA, indicated that protein kinase C may also be involved in the regulation of IGFBP-rP2 gene expression. Although activin A belongs to the TGF-ß superfamily, it did not affect IGFBP-rP2 gene expression in our study.
Because FSH and LH are essential physiological hormones regulating granulosa–luteal cell function, their inhibitory effects on IGFBP-rP2 expression may have a role in follicular growth and development. However, the specific function of IGFBP-rP2 in the ovary is unclear. IGFBP-rP may have IGF-related and/or independent effects in different tissues. IGFBP-rP2 can bind IGF, but with a low affinity compared with the classical IGFBP. Theoretically, gonadotrophin-dependent IGF-II up-regulation (Voutilainen and Miller, 1987; Voutilainen et al., 1996) and IGFBP-rP2 down-regulation would lead to increased IGF bioavailability in developing follicles. However, it looks more likely that direct, IGF-independent effects of IGFBP-rP2 are physiologically more significant than its IGF binding function (Collet and Candy, 1998; Brigstock, 1999). FSH is a granulosa cell mitogen (directly or indirectly) and thus promotes follicular growth. It is also able to stimulate the development of the antral cavity (Strauss and Steinkampf, 1995). The LH surge in pre-ovulatory follicles stimulates a cascade of proteolytic enzymes, including plasminogen activator, plasmin and matrix metalloproteinases. These enzymes bring about the degradation of the perifollicular matrix and, most notably, the decomposition of the meshwork of collagen fibres which provides strength to the follicular wall (Tsafriri and Reich, 1999). Degradation of collagen is therefore a prerequisite for follicular wall weakening and ovulatory rupture (Johnson et al., 1999). Previous studies have shown that IGFBP-rP2 promotes fibroblast collagen synthesis, cell adhesion and cell proliferation. Thus, it may play an important role in tissue regeneration, wound repair and fibrosis (Brigstock, 1999). Therefore, FSH and LH could reduce collagen synthesis indirectly through their inhibitory effect on IGFBP-rP2 expression. The reduced collagen synthesis through decreased IGFBP-rP2 expression could have a role in follicular expansion, antral cavity enlargement and ovulatory rupture. Alternatively, IGFBP-rP2 could promote ovarian cell growth and blood vessel formation, particularly during the FSH-independent pre-antral phase, as previously hypothesized (Wandji et al., 2000; Slee et al., 2001).
In summary, we have found that IGFBP-rP2 mRNA and protein are expressed in human granulosa–luteal cells. The accumulation of IGFBP-rP2 mRNA in the cultured cells was down-regulated by both FSH and LH apparently through protein kinase A, and probably also through protein kinase C pathways. The gonadotrophin-induced inhibition of IGFBP-rP2 expression is likely to have some role in ovarian follicular development and ovulation.
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Ms Merja Haukka and Helena Kemiläinen are thanked for their technical assistance. Mr Olli Horto is thanked for helping with the photographs. Recombinant human FSH and LH for in-vitro experiments were generously provided by Serono-Nordic (Vantaa, Finland), and recombinant human activin A peptide was from Dr A.F.Parlow, NIDDK National Hormone and Pituitary Program, NIH, USA. This study was financially supported by the Emil Aaltonen Foundation, the Jalmari and Rauha Ahokas Foundation, the Paulo Foundation (to J.L.), the Academy of Finland, Paediatric Research Foundation, Novo Nordisk Foundation, and Kuopio University Hospital (to R.V.).
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Notes |
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5 To whom correspondence should be addressed. E-mail: Jiangi.Liu{at}helsinki.fi
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References |
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Adashi, E.Y., Resnick, C.E., Hurwitz, A., Ricciarelli, E., Hernandez, E.R., Roberts, C.T., Leroith, D. and Rosenfeld, R. (1991) Insulin-like growth factors: the ovarian connection. Hum. Reprod., 6, 1213–1219.
Arnheim, N. (1979) Characterization of mouse ribosomal gene fragments purified by molecular cloning. Gene, 7, 83–96.[Web of Science][Medline]
Brigstock, D.R. (1999) The connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family. Endocr. Rev., 20, 189–206.
Collet, C. and Candy, J. (1998) How many insulin-like growth factor binding proteins? Mol. Cell. Endocrinol., 139, 1–6.[Web of Science][Medline]
Devoto, L., Christenson, L.K., McAllister, J.M., Makrigiannakis, A. and Strauss, J.F. III (1999) Insulin and insulin-like growth factor-I and -II modulate human granulosa–lutein cell steroidogenesis: enhancement of steroidogenic acute regulatory protein (StAR) expression. Mol. Hum. Reprod., 5, 1003–1010.
Di Blasio, A.M., Vigano, P. and Ferrari, A. (1994) Insulin-like growth factor-II stimulates human granulosa–luteal cell proliferation in vitro. Fertil. Steril., 61, 483–487.[Web of Science][Medline]
Duncan, M.R., Frazier, K.S., Abramson, S., Williams, S., Klapper, H., Huang, X. and Grotendorst, G.R. (1999) Connective tissue growth factor mediates transforming growth factor ß-induced collagen synthesis: down-regulation by cAMP. FASEB J., 13, 1774–1786.
Giudice, L.C. (1992) Insulin-like growth factors and ovarian follicular development. Endocr. Rev., 13, 641–669.
Hwa, V., Oh, Y. and Rosenfeld, R.G. (1999) The insulin-like growth factor-binding protein (IGFBP) superfamily. Endocr. Rev., 20, 761–787.
Johnson, M.L., Murdoch, J., van Kirk, E.A., Kaltenbach, J.E. and Murdoch, W.J. (1999) Tumour necrosis factor 
regulates collagenolytic activity in preovulatory ovine follicles: relationship to cytokine secretion by the oocyte–cumulus cell complex. Biol. Reprod., 61, 1581–1585.
Kamada, S., Kubota, T., Taguchi, M., Ho, W.R., Sakamoto, S. and Aso, T. (1992) Effects of insulin-like growth factor-II on proliferation and differentiation of ovarian granulosa cells. Horm. Res., 37, 141–149.[Web of Science][Medline]
Kothapalli, D., Hayashi, N. and Grotendorst, G.R. (1998) Inhibition of TGF-ß-stimulated CTGF gene expression and anchorage-independent growth by cAMP identifies a CTGF-dependent restriction point in the cell cycle. FASEB J., 12, 1151–1161.
Liu, J., Heikkilä, P., Kahri, A.I. and Voutilainen, R. (1996) Expression of the steroidogenic acute regulatory protein mRNA in adrenal tumours and cultured adrenal cells. J. Endocrinol., 150, 43–50.
Liu, J., Hydén-Granskog, C. and Voutilainen, R. (2001) Gonadotrophins inhibit expression of inhibin/activin ßB subunit mRNA in cultured human granulosa–luteal cells. Mol. Hum. Reprod., 7, 319–323.
Nakanishi, T., Kimura, Y., Tamura, T., Ichikawa, H., Yamaai, Y., Sugimoto, T. and Takigawa, M. (1997) Cloning of a mRNA preferentially expressed in chondrocytes by differential display-PCR from a human chondrocytic cell line that is identical with connective tissue growth factor (CTGF) mRNA. Biochem. Biophys. Res. Commun., 234, 206–210.[Web of Science][Medline]
Oemar, B.S., Werner, A., Garnier, J.M., Do, D.D., Godoy, N., Nauck, M., Marz, W., Rupp, J., Pech, M. and Luscher, T.F. (1997) Human connective tissue growth factor is expressed in advanced atherosclerotic lesions. Circulation, 95, 831–839.
Schipper, I., Fauser, B.C.J.M., van Gaver, E.B.O., Zarutskie, P.W. and Dahl, K.D. (1993) Development of a human granulosa cell culture model with follicle stimulating hormone responsiveness. Hum. Reprod., 8, 1380–1386.
Slee, R., Hillier, S., Largue, P., Harlow, C.R., Miele, G. and Clinton, M. (2001) Differentiation-dependent expression of connective tissue growth factor and lysyl oxidase messenger ribonucleic acids in rat granulosa cells. Endocrinology, 142, 1082–1089.
Strauss, J.F. III and Steinkampf, M.P. (1995) Pituitary–ovarian interactions during follicular maturation and ovulation. Am. J. Obstet. Gynecol., 172, 726–735.[Web of Science][Medline]
Tsafriri, A. and Reich, R. (1999) Molecular aspects of mammalian ovulation. Exp. Clin. Endocrinol. Diabetes, 107, 1–11.[Web of Science][Medline]
Voutilainen, R. and Miller, W.L. (1987) Coordinate tropic hormone regulation of mRNAs for insulin-like growth factor II and the cholesterol side-chain-cleavage enzyme, P450scc, in human steroidogenic tissues. Proc. Natl. Acad. Sci. USA, 84, 1590–1594.
Voutilainen, R., Tapanainen, J., Chung, B., Matteson, K.J. and Miller, W.L. (1986) Hormonal regulation of P450scc (20,22-desmolase) and P450c17 (17-hydroxylase/17,20-lyase) in cultured human granulosa cells. J. Clin. Endocrinol. Metab., 63, 202–207.
Voutilainen, R., Franks, S., Mason, H.D. and Martikainen, H. (1996) Expression of insulin-like growth factor (IGF), IGF-binding protein, and IGF receptor messenger ribonucleic acids in normal and polycystic ovaries. J. Clin. Endocrinol. Metab., 81, 1003–1008.[Abstract]
Wandji, S.A., Gadsby, J.E., Barber, J.A. and Hammond, J.M. (2000) Messenger ribonucleic acids for MAC 25 and connective tissue growth factor (CTGF) are inversely regulated during folliculogenesis and early luteogenesis. Endocrinology, 141, 2648–2657.
Wang, H.S. and Chard, T. (1999) IGFs and IGF-binding proteins in the regulation of human ovarian and endometrial function. J. Endocrinol., 161, 1–13.[Web of Science][Medline]
Yang, D.H., Kim, H.S., Wilson, E.M., Rosenfeld, R.G. and Oh, Y. (1998) Identification of glycosylated 38-kDa connective tissue growth factor (IGFBP-related protein 2) and proteolytic fragments in human biological fluids, and up-regulation of IGFBP-rP2 expression by TGF-ß in Hs578T human breast cancer cells. J. Clin. Endocrinol. Metab., 83, 2593–2596.
Submitted on July 5, 2001; accepted on October 30, 2001.
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