Mol. Hum. Reprod. Advance Access originally published online on July 8, 2004
Molecular Human Reproduction 2004 10(9):623-628; doi:10.1093/molehr/gah083
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Basic fibroblast growth factor expression in isolated small human ovarian follicles
Department of Anatomy and Structural Biology, School of Medical Sciences, University of Otago, P.O.Box 56, Dunedin, 9001, New Zealand
1 To whom correspondence should be addressed. Email: peter.hurst{at}stonebow.otago.ac.nz
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Abstract |
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Basic fibroblast growth factor (bFGF) is involved in cell proliferation, differentiation, and angiogenesis. It has long been known that bFGF acts as a powerful mitogen for various mammalian granulosa cells in culture. To investigate the possible involvement of bFGF expression in follicle initiation and growth in vivo, we performed nested RT–PCR on ovarian cortical biopsies and quantitative PCR on human follicle populations isolated by laser capture microdissection. Using morphological criteria, follicles were characterized as putative non-growing, primary, or small secondary. RNA was extracted from samples, reverse-transcribed, and relative gene expression levels determined with TaqManTM real-time PCR, using 18S rRNA as the endogenous control. Results confirmed bFGF expression in human adult ovarian cortex, and in the isolated follicles a down-regulation of bFGF mRNA was evident as small follicles develop. This study demonstrates a possible relationship between bFGF mRNA expression and follicle development.
Key words: bFGF/follicle/laser microdissection/ovary/real-time
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Understanding the mechanisms that initiate and control growth of small follicles remains a major challenge in human ovarian biology. The factors and mechanisms involved in this process are yet to be well-defined. Irrespective of gonadotrophin involvement, there is good evidence that local regulatory factors are implicated in this temporal and spatial process.
Basic fibroblast growth factor (bFGF or FGF-2) was first isolated from bovine pituitary in the 1970s (Gospodarowicz, 1975
). It is a ubiquitous molecule that has received sporadic attention in ovarian biology, yet it is known to be a powerful mitogen for granulosa cells of various species in culture (Gospodarowicz and Bialecki, 1978, 1979; Lavranos et al., 1994; Roberts and Ellis, 1999). However, two recent in vitro studies have shown contradictory results. In a whole ovary culture system, the addition of bFGF increased the percentage of growing follicles (Nilsson et al., 2001). In contrast, Derrar et al. (2000) found that bFGF did not change the ratio of growing follicles in an ovarian cortical slice culture system. Unfortunately, these studies did not use robust follicle counting techniques, thus variable follicle distribution in these ovaries was not considered (Hirshfield, 1989).
Immunohistochemical studies have reported the cellular localization of bFGF protein. The overall consensus indicates its presence in oocytes and granulosa cells of most follicle stages, but the degree of immunostaining reported at specific stages is ambiguous (Grothe and Unsicker, 1989; Wordinger et al., 1993; van Wezel et al., 1995; Yamamoto et al., 1997; Berisha et al., 2000). A number of bFGF knockout mice exist (Ortega et al., 1998; Zhou et al., 1998; Montero et al., 2000); all are viable and fertile, but have neuronal, vascular and skeletal defects. These studies did not investigate detailed reproductive function.
Localization of bFGF mRNA within rat ovarian tissue was studied by Guthridge et al. (1992), who performed in situ hybridization on ovary slices at different stages of the oestrous cycle and found that both granulosa and thecal cells of large antral and smaller preantral follicles produced bFGF mRNA. Studies of this molecule in human ovary are sparse; although it has been isolated from human fetal ovaries, IVF granulosa extracts, and cultured IVF granulosa cells (Watson et al., 1992; Di Blasio et al., 1993; Yeh and Osthanondh, 1993). This study was undertaken to investigate bFGF gene expression in adult human ovarian cortex and to quantify bFGF gene expression in isolated small follicles of morphologically distinct stages.
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Materials and methods |
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Tissue collection
Ovarian biopsies were collected from seven healthy fertile women (26–34 years of age) undergoing tubal ligation for fertility control. Informed consent was obtained from all women and the work approved by the Otago Ethics Committee (reference no: 99/12/113). Wedges of ovary measuring
0.5 cm3 were halved; one half was immersed in RNAlaterTM (Ambion Inc., USA) and refrigerated for subsequent RNA extraction, the other half was immediately embedded in O.C.T. cryopreservation compound (Tissue-Tek; Miles, Inc., USA) and stored at –80°C.
RNA extraction and RT of whole biopsies
Biopsies were removed from RNAlaterTM and the surface epithelium and medullary tissue dissected with a scalpel under a dissecting microscope. The resultant cortical tissue was finely chopped and added to TRIzol® reagent (Invitrogen, USA). To break apart the heavy collagen matrix, specimens were thoroughly homogenized and sonicated. Total RNA was isolated according to standard TRIzol® procedures; RNA was precipitated, washed in 75% ethanol, then dissolved in ribonuclease (RNase)-free Tris–EDTA (pH 8.0). RNA quality was assessed by spectrophotometry and agarose gel electrophoresis. Total RNA (64 ng) was reverse-transcribed with SuperScriptTM II first strand synthesis kit (Invitrogen) using oligo deoxythymidine as primer.
Nested PCR and DNA sequencing
All primers were designed for human bFGF from GenBank sequence NM_002006.2. First round PCR was performed using cDNA equivalent of 10 ng mRNA, 2 IU Platinum® Taq DNA polymerase (Invitrogen), and the outer primers: forward 5'-TCTTCCTGCGCATCCACC-3' and reverse 5'-TCAGCTCTTAGCAGACATTGGAAGA-3' in a 50 µl reaction according to manufacturer's protocol. Cycling parameters consisted of an initial denaturing step at 94°C for 2 min, then 30 cycles at 94°C for 20 s, 58°C for 30 s, and 72°C for 30 s. An extension step of 72°C for 5 min was added at the end of amplification.
Two microlitres of first round product was used as template for another 30 cycles of PCR with the inner primers: forward 5'-TGTGCTAACCGTTACCTGGCT-3' and reverse 5'-CAGTGCCACATACCAACTG-3'. A 50 µl reaction was performed according to the manufacturer's specifications. Cycling parameters were the same as above except that an annealing temperature of 59°C was used.
Nested PCR products were visualized by agarose gel electrophoresis. Products were purified using the Qiagen PCR purification kit (Qiagen, Canada), and sequenced (ABI377 DNA sequencer; Applied Biosystems, USA).
Laser capture microdissection (LCM)
LCM is a method of isolating specific cell populations from tissue sections using a laser mounted on an inverted research microscope (PixCell® II; Arcturus, USA). Sections (8 µm) were cut at –20°C from O.C.T.-embedded biopsies and mounted on RNase-free slides. After brief fixation in 70% ethanol, sections were stained with Histogene staining solution (Arcturus), and dehydrated to xylene. Slides were stored in desiccated containers until LCM.
Before isolation, follicles present in the section were microscopically classified using morphological criteria similar to those described by Gougeon (1996). Human primordial and transitory follicles were categorized by the presence of squamous granulosa cells and subsequently classed as putative non-growing follicles; when the oocyte was clearly surrounded by a complete layer of cuboidal granulosa cells, the follicle was classed as a primary follicle. Small secondary follicles had more than one layer of granulosa cells.
Laser pulses were fired at oocytes and granulosa cells of selected follicles; the disposable tissue collection cap (High-sensitivity caps, Arcturus) and micro-dissected tissue were lifted away from the section (Figure 1). The physical size limitations of the laser beam precluded the dissection of individual follicle cells. Due to the small amount of tissue (and thus mRNA), LCM-harvested follicle cells were pooled for each follicle type from each individual patient.
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RNA extraction and RT of isolated follicles
Captured tissue was incubated in PicoPureTM extraction buffer (Arcturus) at 42°C for 30 min. Total RNA was isolated using the PicoPureTM RNA isolation kit (Arcturus) according to the manufacturer's instructions. As the total RNA extracted from these samples was extremely limited, no attempt was made to quantify the amount of RNA in the sample. Consequently, the entire sample was used in subsequent RT reactions. RT was undertaken as above, except that random hexamers were used as primers.
Comparative CT method for relative quantification of gene expression
Duplicate multiplex reactions were carried out on follicle samples using the ABI TaqManTM bFGF primer/probe set with FAM dye labelling the probe (Assay-on-DemandTM Hs00266645_m1; Applied Biosystems). The normalizer 18S was used, labelled with the fluorescent dye VIC (Pre-developed TaqManTM Assay; Applied Biosystems). Primer concentrations were pre-limited for multiplex reactions.
Thirty microlitre reactions were prepared in 96-well optical reaction plates with optical adhesive covers (ABI PrismTM Applied Biosystems) using TaqManTM Universal PCR Master Mix (Applied Biosystems). Using the ABI Prism® 7000 (Applied Biosystems), samples were heated to 50°C for 2 min, then 95°C for 10 min before 40 cycles at 95°C for 15 s and 60°C for 1 min as per manufacturer's instructions.
Real-time data were analysed using the comparative CT method: where CT is the cycle number at which the fluorescence reading is first recorded above background levels (Bustin, 2000). The comparative CT method is similar to the standard curve method, except that it uses the arithmetic formula, 2–
CT to achieve relative quantification (for detailed information see Applied Biosystems' User Bulletin #2). A prior validation experiment was performed to demonstrate that amplification efficiencies of bFGF and 18S primer/probe sets were approximately equal: efficiencies were within 0.1 (data not shown).
Evaluation of sample specificity
To confirm purity of LCM isolates, separate samples of ovarian stromal cells were isolated via LCM. RNA extraction and RT was performed as for follicle cell preparations. Sample purity was confirmed by standard PCR with primers for FSH receptor and cytochrome P450 17
-hydroxylase (P450c17). Primer details and annealing temperatures for PCR are summarized in Table I.
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Statistical analysis of comparative CT method for relative quantification
As multiplex reactions occur in the same tube, raw bFGF CT data are firstly normalized by subtracting the corresponding 18S CT. Duplicates are then averaged to give the mean
CT. The comparative CT method is a relative measure of mRNA expression, thus one of the samples can be used as a calibrator. Here the putative non-growing follicle samples were chosen to be the calibrator for each individual. Subtracting the CT for putative non-growing follicles from the CT of all samples gave the relative change (CT). It is important to note that relative comparisons were only made between follicle types within the same patient (intra-personal variation).
Slopes calculated from the regression line for each patient were used in a one-sample t-test, testing against zero.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Confirmation of bFGF gene expression in human cortical extracts
To study expression of bFGF in human ovary cortical extracts, nested RT–PCR was performed. This resulted in a product of the expected 150 bp length (Figure 2a, lane 3). Sequencing confirmed that the 150 bp product corresponded to the appropriate part of GenBank sequence NM_002006.2 (Figure 2b). This fragment crosses an intron/exon boundary and thus established that the product was mRNA in origin. The fact that nested PCR was required to detect bFGF indicated that expression of this gene was low.
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) and inner primers (
) cross multiple intron/exon boundaries. The black box represents the 150 bp amplicon; numbers indicate nucleotide position for beginning and end of amplicon in relation to GenBank sequence Relative quantification of bFGF gene expression across follicle stages
Specific follicle populations were isolated from ovarian biopsies from seven women using LCM. Due to the nature of human material each biopsy contained variable populations of follicle types, and thus for every patient studied different follicle types were isolated (summarized in Table II).
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Table III shows the results from the real-time PCR for these samples. All seven patients showed a down-regulation of bFGF expression across early follicle development.
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To demonstrate these trends, the mean of individual slopes for each patient is presented in Figure 3.
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Confirmation of sample specificity
To test the specificity of our LCM method, four different genes were used as cell markers (Figure 4). ß-Actin (750 bp) and bFGF (150 bp) were found in all the RT + samples (cortical extract, oocyte/granulosa cells from small follicles, and stroma). FSH receptor (340 bp; granulosa cell marker) was detected in ovarian cortex and the small follicle sample, but not in the stromal LCM sample, indicating that the small follicle sample contained granulosa cells. P450c17 (418 bp; an enzyme found in steroid-producing cells such as thecal or stromal tissue) was detected in ovarian cortex, and the stromal sample. The small follicle sample was negative for P450c17, demonstrating that surrounding stromal material did not contaminate preparations of oocytes and granulosa cells.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this study, putative non-growing, primary, and small secondary follicles have, for the first time, been isolated by laser microdissection. This isolation technique provides an opportunity to study gene expression at specific stages of follicle development from snap-frozen human biopsies. We report here that bFGF was not only expressed by adult ovarian cortex, but that bFGF mRNA was present in small follicles and expression decreased over early follicle development.
bFGF gene expression has been reported in human fetal ovaries (Yeh and Osathanondh, 1993) and we confirm bFGF mRNA expression in healthy adult ovarian cortex. In earlier studies, there was uncertainty regarding the detection of bFGF transcripts (Neufeld et al., 1987; Koos and Olson, 1989; Stirling et al., 1991), but with technical advances it is now evident that bFGF is expressed in cultured and freshly prepared granulosa cells of large follicles from many different species (Guthridge et al., 1992; Watson et al., 1992; Berisha et al., 2000; Shimizu et al., 2002). Here, in the human, we have shown that small ovarian follicles express the gene for bFGF.
By using real-time PCR, we not only demonstrated bFGF mRNA expression in the smallest follicles, but also compared relative expression levels across early follicle growth. All seven patients showed the same decreasing trend in bFGF mRNA expression. The only other study to investigate bFGF mRNA expression across early follicle development used in situ hybridization, which localized the mRNA for bFGF in granulosa cells and theca cells of rat preantral follicles (Guthridge et al., 1992). Similarly, here we report bFGF mRNA in small follicles in human specimens and further suggest a down-regulation of the mRNA during early follicle growth.
The LCM isolation technique has allowed the clear separation of granulosa cells and oocytes from surrounding theca or stromal tissue with minimum manipulation. The purity of LCM sample preparation was tested using standard RT–PCR. The detection of FSH receptor and P450c17 in follicle and stromal samples respectively indicates that laser microdissection precisely isolated follicle samples from ovarian sections. This technique is especially useful in human material as the adult ovary is extremely collagenous and sparsely populated with follicles. Alternative small follicle isolation techniques would be impractical for gene expression studies unless undesirable mechanical or protease treatments as well as extended incubations are used (Oktay et al., 1997).
A number of immunohistochemical studies reported high levels of bFGF protein in the oocyte (van Wezel et al., 1995; Nilsson et al., 2001), whereas others were unable to detect bFGF in oocytes at all (Wordinger et al., 1993). This highlights the variability inherent in immunodetection. We would have liked to use LCM to clarify whether bFGF mRNA was found in oocytes or granulosa cells, but due to the physical limitations of the laser, more detailed dissection of the follicle (i.e. oocytes as distinct from granulosa cells) was considered unreliable and not performed. To investigate cellular sources of bFGF mRNA further, in situ hybridization on human ovarian biopsies is currently in progress.
It is interesting to speculate on the role of bFGF in follicle development and, in light of these findings, what its specific function might be in small follicles. Many groups have shown that bFGF has angiogenic properties (Bikfalvi et al., 1997); but since very small follicles have no direct blood supply it is unlikely that the relatively increased levels of bFGF mRNA in the putative non-growing follicles reported here relate to angiogenesis. bFGF has been previously implicated in inhibitory interactions with the gonadotrophin receptors LH and FSH (Mondschein and Schomberg, 1981; Biswas et al., 1988; Yamoto et al., 1993; Kanzaki et al., 1994). The down-regulation of bFGF message in early follicle development reported here provides evidence for bFGF as an inhibitor of follicle growth through attenuation of gonadotrophin receptor induction. To date there is little direct or convincing evidence for apoptosis in primordial follicles; our finding of higher levels of bFGF mRNA in putative non-growing follicles supports the hypothesis that bFGF could inhibit apoptosis (Lynch et al., 2000). Our results indicate a role for bFGF in small human ovarian follicles that requires further investigation.
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Acknowledgements |
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The authors wish to thank Associate Professor Wayne Gillett, Mr Adel Mekhail, and Dr Sarah Tout for assistance in obtaining biopsy samples, and Dr Melanie Bell for statistical assistance. Work supported by the Department of Anatomy and Structural Biology. J.H.Q. was the recipient of a University of Otago Postgraduate Scholarship.
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References |
|---|
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Berisha B, Schams D, Kosmann M, Amselgruber W and Einspanier R (2000) Expression and localisation of vascular endothelial growth factor and basic fibroblast growth factor during the final growth of bovine ovarian follicles. J Endocrinol 167, 371–382.[Abstract]
Bikfalvi A, Klein S, Pintucci G and Rifkin DB (1997) Biological roles of fibroblast growth factor-2. Endocr Rev 18, 26–45.
Biswas SB, Hammond RW and Anderson LD (1988) Fibroblast growth factors from bovine pituitary and human placenta and their functions in the maturation of porcine granulosa cells in vitro. Endocrinology 123, 559–566.
Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25, 169–193.[Abstract]
Derrar N, Price CA and Sirard MA (2000) Effect of growth factors and co-culture with ovarian medulla on the activation of primordial follicles in explants of bovine ovarian cortex. Theriogenology 54, 587–598.[CrossRef][Web of Science][Medline]
Di Blasio AM, Vigano P, Cremonesi L, Carniti C, Ferrari M and Ferrari A (1993) Expression of the genes encoding basic fibroblast growth factor and its receptor in human granulosa cells. Mol Cell Endocrinol 96, R7–R11.[CrossRef][Web of Science][Medline]
Gospodarowicz D (1975) Purification of a fibroblast growth factor from bovine pituitary. J Biol Chem 250, 2515–2520.
Gospodarowicz D and Bialecki H (1978) The effects of the epidermal and fibroblast growth factors on the replicative lifespan of cultured bovine granulosa cells. Endocrinology 103, 854–865.
Gospodarowicz D and Bialecki H (1979) Fibroblast and epidermal growth factors are mitogenic agents for cultured granulosa cells of rodent, porcine, and human origin. Endocrinology 104, 757–764.
Gougeon A (1996) Regulation of ovarian follicular development in primates: facts and hypotheses. Endocr Rev 17, 121–155.
Grothe C and Unsicker K (1989) Immunocytochemical localization of basic fibroblast growth factor in bovine adrenal gland, ovary, and pituitary. J Histochem Cytochem 37, 1877–1883.[Abstract]
Guthridge M, Bertolini J, Cowling J and Hearn MT (1992) Localization of bFGF mRNA in cyclic rat ovary, diethylstilbesterol primed rat ovary, and cultured rat granulosa cells. Growth Factors 7, 15–25.[Medline]
Hirshfield AN (1989) Granulosa cell proliferation in very small follicles of cycling rats studied by long-term continuous tritiated-thymidine infusion. Biol Reprod 41, 309–316.[Abstract]
Kanzaki M, Hattori M, Horiuchi R and Kojima I (1994) Basic fibroblast growth factor induces luteinizing hormone receptor expression in the presence of insulin-like growth factor-I in ovarian granulosa cells. Mol Cell Endocrinol 101, 95–99.[CrossRef][Web of Science][Medline]
Koos RD and Olson CE (1989) Expression of basic fibroblast growth factor in the rat ovary: detection of mRNA using reverse transcription–polymerase chain reaction amplification. Mol Endocrinol 3, 2041–2048.
Lavranos TC, Rodgers HF, Bertoncello I and Rodgers RJ (1994) Anchorage-independent culture of bovine granulosa cells: the effects of basic fibroblast growth factor and dibutyryl cAMP on cell division and differentiation. Exp Cell Res 211, 245–251.[CrossRef][Web of Science][Medline]
Lynch K, Fernandez G, Pappalardo A and Peluso JJ (2000) Basic fibroblast growth factor inhibits apoptosis of spontaneously immortalized granulosa cells by regulating intracellular free calcium levels through a protein kinase Cdelta-dependent pathway. Endocrinology 141, 4209–4217.
Mondschein JS and Schomberg DW (1981) Growth factors modulate gonadotropin receptor induction in granulosa cell cultures. Science 211, 1179–1180.
Montero A, Okada Y, Tomita M, Ito M, Tsurukami H, Nakamura T, Doetschman T, Coffin JD and Hurley MM (2000) Disruption of the fibroblast growth factor-2 gene results in decreased bone mass and bone formation. J Clin Invest 105, 1085–1093.[Web of Science][Medline]
Neufeld G, Ferrara N, Schweigerer L, Mitchell R and Gospodarowicz D (1987) Bovine granulosa cells produce basic fibroblast growth factor. Endocrinology 121, 597–603.
Nilsson E, Parrott JA and Skinner MK (2001) Basic fibroblast growth factor induces primordial follicle development and initiates folliculogenesis. Mol Cell Endocrinol 175, 123–130.[CrossRef][Web of Science][Medline]
Oktay K, Briggs D and Gosden RG (1997) Ontogeny of follicle-stimulating hormone receptor gene expression in isolated human ovarian follicles. J Clin Endocrinol Metab 82, 3748–3751.
Ortega S, Ittmann M, Tsang SH, Ehrlich M and Basilico C (1998) Neuronal defects and delayed wound healing in mice lacking fibroblast growth factor 2. Proc Natl Acad Sci USA 95, 5672–5677.
Roberts RD and Ellis RC (1999) Mitogenic effects of fibroblast growth factors on chicken granulosa and theca cells in vitro. Biol Reprod 61, 1387–1392.
Shimizu T, Jiang JY, Sasada H and Sato E (2002) Changes of messenger RNA expression of angiogenic factors and related receptors during follicular development in gilts. Biol Reprod 67, 1846–1852.
Stirling D, Waterman MR and Simpson ER (1991) Expression of mRNA encoding basic fibroblast growth factor (bFGF) in bovine corpora lutea and cultured luteal cells. J Reprod Fertil 91, 1–8.
van Wezel IL, Umapathysivam K, Tilley WD and Rodgers RJ (1995) Immunohistochemical localization of basic fibroblast growth factor in bovine ovarian follicles. Mol Cell Endocrinol 115, 133–140.[CrossRef][Web of Science][Medline]
Watson R, Anthony F, Pickett M, Lambden P, Masson GM and Thomas EJ (1992) Reverse transcription with nested polymerase chain reaction shows expression of basic fibroblast growth factor transcripts in human granulosa and cumulus cells from in vitro fertilisation patients. Biochem Biophys Res Commun 187, 1227–1231.[CrossRef][Web of Science][Medline]
Wordinger RJ, Brun-Zinkernagel AM and Chang IF (1993) Immunohistochemical localization of basic fibroblast growth factor (bFGF) within growing and atretic mouse ovarian follicles. Growth Factors 9, 279–289.[Web of Science][Medline]
Yamamoto S, Konishi I, Nanbu K, Komatsu T, Mandai M, Kuroda H, Matsushita K and Mori T (1997) Immunohistochemical localization of basic fibroblast growth factor (bFGF) during folliculogenesis in the human ovary. Gynecol Endocrinol 11, 223–230.[Web of Science][Medline]
Yamoto M, Shikone T and Nakano R (1993) Opposite effects of basic fibroblast growth factor on gonadotropin-stimulated steroidogenesis in rat granulosa cells. Endocr J 40, 691–697.[Web of Science][Medline]
Yeh J and Osathanondh R (1993) Expression of messenger ribonucleic acids encoding for basic fibroblast growth factor (FGF) and alternatively spliced FGF receptor in human fetal ovary and uterus. J Clin Endocrinol Metab 77, 1367–1371.[Abstract]
Zhou M, Sutliff RL, Paul RJ, Lorenz JN, Hoying JB, Haudenschild CC, Yin M, Coffin JD, Kong L, Kranias EG et al. (1998) Fibroblast growth factor 2 control of vascular tone. Nat Med 4, 201–207.[CrossRef][Web of Science][Medline]
Submitted on May 17, 2004; accepted on June 9, 2004.
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