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a Division of Reproductive Biology, Department of Gyn/Ob, Stanford University Medical School, Stanford, California 94305-5317
b Department of Ob/Gyn, University of California School of Medicine, Los Angeles, California 90095
c Department of Animal Sciences, University of Illinois, Urbana, Illinois 61801
d Institut National de la Sante et de la Recherche Medicale, U-355 Clamart, France
e Department of Obstetrics, Gynecology and Cell Biology, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gene promoter. We investigated the pattern of WT1 expression in a wide variety of species and also over the reproductive life span in rats. In chicken ovaries, Northern blot analysis revealed the presence of WT1 transcript in small healthy white follicles (1–5 mm in diameter) and its absence in small yellow (6–12 mm in diameter) or larger follicles (F1–F5). In pig and monkey ovaries, WT1 expression was limited to granulosa cells of preantral follicles, as shown by in situ hybridization analysis. In rats, Northern blot analyses demonstrated the presence of WT1 transcript in the ovaries of young (3-mo-old) and middle-aged (9-mo-old) rats on the proestrous day, with a decrease in old (12-mo-old) rats in persistent estrus. In situ hybridization analysis further suggested that the decrease in WT1 expression in aging ovaries was associated with fewer immature follicles. Thus, WT1 expression is restricted to immature follicles in diverse avian and mammalian species and over the reproductive life span in rats. These data demonstrated that WT1 is a marker for immature follicles and suggested a potential role of this transcriptional repressor in the slow growth of early follicles.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The WT1 gene, first discovered because of its deletion in a small subset of patients with Wilms' tumors, encodes a transcription factor with zinc fingers and shares homology with proteins of the early growth response (EGR) gene family [3]. WT1 expression is restricted to a limited set of tissues, including the kidney and gonads [4, 5]. In the kidney, the WT1 protein has been shown to function as a transcriptional repressor of growth-related genes [6]. In contrast, little is known concerning the function and regulation of WT1 in gonads. WT1 mRNA is found in granulosa cells of ovarian follicles and Sertoli cells of the testis [4, 5]. It has also been reported that development of the gonadal ridge is abnormal in WT1-deficient mice [7], implicating an important role for WT1 in embryonic gonad development. Additionally, some patients with Denys-Drash Syndrome (genitourinary malformation and gonadal dysgenesis) have mutations in the WT1 locus, suggesting that it also has a role in gonadal development in the primate.
Recent studies have focused on the potential role of WT1 in the regulation of follicular growth. In ovaries of prepubertal rats, WT1 mRNA was exclusively localized to immature ovarian follicles; furthermore, WT1 protein repressed the promoter activity of inhibin-, a gene involved in follicle differentiation [8]. These data suggest a role for WT1 in the slowing of early follicular growth. However, direct interaction between the WT1 protein and the inhibin- promoter has not been demonstrated. Moreover, the expression pattern of WT1 in the ovaries of different species, as well as the expression patterns of WT1 over the female reproductive life span, have not been evaluated. In the present study, a gel retardation assay was used to determine whether recombinant WT1 protein binds to inhibin- promoter. The pattern of WT1 mRNA expression was also evaluated in the ovaries of diverse mammalian and avian species and during reproductive senescence in rat.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human CG (CR-127; 14,900 IU/mg) was obtained from the National Hormone and Pituitary Distribution Program, NIDDK, NIH (Baltimore, MD). The following reagents were purchased from the indicated sources: eCG (Calbiochem, La Jolla, CA), diethylstilbestrol; Sigma, St. Louis, MO), the riboprobe system (Promega, Madison, WI), restriction enzymes (Boehringer, Mannheim, Germany), [-32P]CTP and [32P]
-ATP (Amersham, Arlington Heights, IL), and [-35S]UTP (NEN Dupont, Boston, MA).
Animals and Tissue Preparation
Avian ovaries were collected from Single Comb White Leghorn hens, 7–12 mo of age, with regular sequences of at least 8 eggs. Follicles were then removed according to the following stages of development: 1) F1-F5, which represent the five largest follicles in the order of expected ovulation time (9); 2) the small healthy white follicles (1–5 mm in diameter); 3) the small healthy yellow follicles (6–12 mm in diameter); and 4) the small atretic yellow follicles [9]. Theca and granulosa cells were isolated from all but the small yellow follicles as previously described [10]. Tissues were immediately frozen until RNA extraction.
Porcine ovarian tissue from a mature animal was obtained from a local abattoir and placed for 10–15 min in ice-cold 0.15 M NaCl for subsequent follicle isolation. Follicles ranging from 3–7 mm in diameter [11] were randomly dissected within 2–3 h of ovary collection, and fixed for in situ hybridization analysis.
Monkey (Macaca fascicularis) ovaries were obtained from two normally cycling adults undergoing oophorectomy for other research protocols. Small portions of ovary were fixed in 4% paraformaldehyde for 6 h, washed in phosphate buffer, and then placed in 0.5 M sucrose overnight. The tissues were then frozen and stored for in situ hybridization analysis.
Young virgin (3 mo of age) and middle-aged retired breeder (8 mo of age) Long-Evans rats were obtained from Charles River Laboratories (Portage, MI). Daily vaginal smears were taken from these animals to determine their estrous cycle pattern, and rats that had exhibited at least three consecutive 4-day cycles were used for the experiments. Old (12 mo of age) females that had displayed more than 15 consecutive days of cornified vaginal cytology were classified as persistently estrous [12]. On the morning (1100 h) of proestrus or during persistent estrus, the animals were killed by cervical dislocation, and ovaries were collected for Northern blot and in situ hybridization analyses.
Gel Retardation Assays
Recombinant WT1 protein with six additional histidine residues attached at its amino-terminus was produced in Escherichia coli and purified by nickel chelate chromatography as previously described [13]. Gel retardation assays were performed using radiolabeled oligonucleotides containing a putative WT1 binding site corresponding to nucleotides -164 to -144 of the rat inhibin- promoter [8]. Oligonucleotides were labeled by filling in overhanging linker ends with [32P]-ATP using T4 polynucleotide kinase. Binding conditions were as follows: 10 mM HEPES, pH 7.9; 5 mM Tris-HCl, pH 7.5; 50 mM NaCl; 1 mM EDTA; 1 mM dithiothreitol; 10% glycerol; 2 µg of poly(dI-dC); 0.5 ng of 104 cpm end-labeled DNA oligonucleotide probe; and the recombinant WT1 protein. For the competition studies, a molar excess of unlabeled competitor oligonucleotide (inhibin- promoter oligomer or unrelated Oct2A oligomer) was added to the binding reaction. Supershifts were performed using anti-WT1 polyclonal sera (Santa Cruz Biotechnology, Santa Cruz, CA). The DNA-protein binding complexes were loaded onto a 5% polyacrylamide gel (39:1 acrylamide-bisacrylamide) and run in 0.5-strength TBE (50 mM Tris, 50 mM boric acid, and 1 mM EDTA, pH 8.0) for 2–3 h at 8 V/cm. The gels were dried and autoradiographed using Kodak XAR-5 films (Eastman Kodak, Rochester, NY).
Preparation of RNA Probes
A 656-basepair DNA fragment of rat WT1 cDNA corresponding to the region coding for the zinc finger domain of WT1 [5] was subcloned into a pSK vector (Promega). The plasmid was linearized with EcoRI and transcribed with T7 RNA polymerase to generate an antisense RNA probe, or linearized with HindIII and transcribed with T3 RNA polymerase to generate a sense RNA probe. The probes were labeled with [32P]CTP or [35S]UTP for Northern blot or for in situ hybridization analyses using the riboprobe in vitro transcription system (Promega).
Northern Blot Hybridization
Ovarian tissues were homogenized in Tri Reagent solution (Molecular Research Center Inc., Cincinnati, OH) to extract total RNA [8]. For Northern blot analysis, 10–20 µg total RNA were fractionated by electrophoresis on 1.0% agarose-formaldehyde gels, transferred to nylon membranes by capillary blotting with 20-strength sodium citrate-sodium chloride (SSC; (single-strength SSC = 0.15 M sodium chloride, 0.015 M sodium citrate), and covalently cross-linked to the membranes using a UV cross-linker (Stratagene, La Jolla, CA). Membranes were prehybridized for 6 h at 65°C in a solution containing 50% formamide, 5-strength SSC, 1.6-strength Denhardt's solution, 1 mM EDTA, 250 µg/ml denatured herring sperm DNA, and 500 µg/ml yeast tRNA. Hybridization was carried out at 65°C overnight in the same solution containing 1 x 106 cpm/ml of the [32P]-labeled rat WT1 cRNA probe. After hybridization, membranes were washed twice in double-strength SSC and 0.1% SDS for 10 min at room temperature, and then washed 2–3 times in 0.1-strength SSC and 0.1% SDS at 65°C for 10 min. Membranes were then exposed onto Fuji x-ray films (Fuji Photo Film, Minami-Ashigarashi, Japan) for 2–3 days at -70°C.
In Situ Hybridization Analysis
Ovarian tissues were fixed at 4°C for 6 h in 4% paraformaldehyde in PBS, and then immersed in 0.5 M sucrose in PBS overnight. Cryostat sections (10–14 mm thick) were mounted on poly-L-lysine-coated microscope slides, fixed in 4% paraformaldehyde in PBS, and stored at -70°C until analyzed. The hybridization procedure was essentially the same as previously described [8]. In brief, sections were pretreated sequentially with 0.2 M HCl, double-strength SSC, pronase (0.125 mg/ml), 4% paraformaldehyde, and acetic anhydride in triethanolamine. Hybridization was carried out at 50°C overnight in a mixture containing the labeled riboprobe (108 cpm/ml), 50% formamide, 0.3 M NaCl, 10 mM Tris-HCl, 5 mM EDTA, single-strength Denhardt's solution, 10% dextran sulfate, 1 mg/ml carrier transfer RNA, and 10 mM dithiothreitol. Post-hybridization washing included ribonuclease (RNase) A (25 µg/ml) treatment at 37°C for 30 min and a final stringency of 0.1-strength SSC. Slides were dipped into NTB-2 emulsion (Eastman Kodak) and exposed at 4°C until development at 3–4 wk later. Sections were stained with hematoxylin and eosin and examined under the light microscope with bright- and darkfield illumination.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Promoter
We have previously demonstrated that, in transfected cells, overexpression of WT1 represses the promoter activity of the inhibin- gene [8]. To further confirm that the WT1 protein interacts directly with the inhibin- promoter, gel retardation assays were performed with the zinc-finger containing recombinant WT1 protein produced in bacteria. As shown in Figure 1, the WT1 protein bound to the oligonucleotide probe spanning nucleotides -164 to -144 of the inhibin- promoter as demonstrated by a shift of the labeled probe to a high-molecular-weight region. In the presence of excess unlabeled inhibin- oligomer, this WT1 DNA-protein complex was decreased in a dose-dependent manner. In contrast, a nonrelated oligomer, Oct2A, did not alter the binding of WT1 to the labeled inhibin- promoter probe. Furthermore, this complex between the WT1 protein and the inhibin- promoter DNA was supershifted after the addition of the anti-WT1 antibody, resulting in the formation of a complex of larger molecular weight. Thus, gel retardation assays suggested that a high-affinity WT1 binding site was present in the inhibin- promoter.
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Expression of WT1 mRNA in Avian Ovaries
We took advantage of the well-defined follicle hierarchy in avian ovaries and the ease of obtaining large numbers of cells for mRNA analysis to determine whether restricted WT1 mRNA expression occurs in avian ovaries as demonstrated in immature rats [8]. As shown in Figure 2, Northern blot analysis revealed that a major WT1 transcript of 3.7 kilobases (kb) was detectable only in small white follicles (1–5 mm in diameter). Granulosa or theca cells from small yellow (6–12 mm in diameter) and larger follicles (F1 to F5) showed negligible hybridization signals. These findings suggest that WT1 expression in avian ovaries, like rodent ovaries, is restricted to immature follicles.
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Expression of WT1 mRNAs in Porcine and Monkey Ovaries
We further performed in situ hybridization analysis of WT1 mRNA in porcine ovarian fragments. As shown in Figure 3, A and B, a preovulatory follicle collected from mature porcine ovaries had several immature follicles in its vicinity. These primary and secondary follicles as well as the surface epithelium expressed WT1 mRNA, whereas the large preovulatory follicle did not (Fig. 3, A and B). Under higher magnification, only granulosa cells of immature preantral follicles exhibited WT1 mRNA signals, with lower signals in theca cells (Fig. 3, C–E). In contrast, studies using a sense WT1 probe showed no hybridization signals (Fig. 3F).
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The restricted pattern of WT1 expression was also found in monkey ovary. Granulosa cells of secondary (2F) and preantral follicles (PAF) exhibited strong signals, whereas small antral follicles exhibited weaker signals (Fig. 4, A–D). In contrast, atretic (Atr) and large antral follicles (AF) exhibited negligible WT1-mRNA signals (Fig. 4, A and B). Nonspecific signals could be detected in hemosiderin pigment derived from red blood cells in sections hybridized with antisense (Fig. 4, A–D) and sense probe (Fig. 4, E and F).
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Expression of WT1 mRNAs in Ovaries of Adult and Aging Rats
To further examine the restricted distribution of WT1 mRNA at different stages of reproductive life, ovaries of adult and aging rats were examined. Northern blot analyses revealed the presence of a 3.7-kb WT1 transcript in ovaries of young proestrous rats at 3 mo of age (Fig. 5; YP). Comparable levels of WT1 mRNA was found in the ovaries of middle-aged rats at 9 mo of age (Fig. 5; MP), whereas it was decreased in ovaries of persistently estrous rats at 12 mo of age (Fig. 5; PE; 48.5% decrease as compared with the MP group, n = 3).
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Ovaries of young proestrous rats contained numerous developing follicles at different stages of growth. In situ hybridization analysis showed that WT1 mRNA was limited to granulosa cells of healthy secondary and preantral follicles as well as the surface epithelium, but was not expressed in antral and preovulatory follicles or in corpus luteum (Fig. 6, A and B). Likewise, ovaries of middle-aged proestrous rats contained many growing follicles at all stages of development as well as corpora lutea (Fig. 6C), and the WT1 signal was again localized to both surface epithelium and small follicles (Fig. 6, C and D). In contrast, ovaries of older persistently estrous rats contained few preantral and healthy growing follicles but numerous enlarged cystic follicles with attenuated granulosa layers (Fig. 6E). In situ hybridization analysis revealed high levels of WT1 gene expression only in small primary follicles (Fig. 6, E and F). Under higher magnifications, the WT1 mRNA signals were limited to the granulosa cells of preantral follicles in ovaries from persistently estrous rats (Fig. 6G). Hybridization with sense WT1 probe verified the specificity of signals (Fig. 6H).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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promoter, and that the restricted pattern of WT1 expression in immature ovarian follicles is conserved in chickens and three mammalian species, and maintained throughout the reproductive life span in rats. Although the regulation of antral follicle development during the final phases of follicle maturation has been extensively studied, the development of preantral follicles remains largely unexplored, partially because of the lack of biochemical markers for the immature follicles. The present study demonstrates that WT1 is a useful marker for the analysis of early follicle development in diverse species throughout the reproductive life span.
A repressor role for WT1 in the regulation of the inhibin- gene is consistent with the present finding demonstrating a direct binding of the WT1 protein to its consensus binding sequence present in the inhibin- promoter. It has been shown that WT1 protein binds to different consensus sites similar to the Egr-1/WTE site [13, 14]. In the proximal sequence of the inhibin- promoter, there is a potential binding sequence (-158 to -149, GAGTGGGAGA) that shares high homology with the WTE binding site [14]. The present study demonstrated that WT1 protein indeed interacts with a putative WT1 binding site in the inhibin- promoter, and thus confirms and extends the previous finding showing the repression of inhibin- promoter activity by WT1 [8]. It is, however, important to note that inhibin- is expressed in both granulosa and theca cells of preantral follicles, whereas WT1 is expressed exclusively in granulosa cells.
Chickens, rats, pigs, and monkeys all show similar expression patterns of WT1 within the ovary. All of these species have a limited pool of follicles established very early in life and exhibit limited reproductive life spans presumably determined by the depletion of follicles in the ovary. The expression pattern of WT1 in immature follicles is uniform throughout these species. The early generations of follicle growth are exceedingly protracted, but the rate of granulosa cell proliferation increases with increasing follicle size. In the rat, for instance, the first three generations of follicle growth takes longer than those of the remaining seven generations [15, 16]. In contrast, follicles grow more rapidly during the final stages of development at the time of antrum formation (eighth generation) than at any other time during development. Likewise, in cattle and humans, the initial stages of follicle growth take several months, whereas the final stages of growth are accomplished in several days [17–19]. In the present studies, WT1 expression was prominent in small follicles, but decreased when follicle size was increased, implicating WT1 in the slowing of granulosa cell development at the early stages of follicle growth.
It has been suggested that the growth of small preantral follicles may be regulated by gonadotropins [1, 20–23] and local growth factors [24, 25]. It is therefore possible that WT1 delays follicle growth by repressing the transcriptional activity of genes encoding gonadotropin receptors and intraovarian growth factors, or their receptors. Indeed, WT1 has been shown to regulate several growth factors including transforming growth factor ß (TGFß), platelet-derived growth factor, insulin-like growth factor II (IGF-II), and IGF-I receptor in other systems [6]. The physiological function of WT1 in relation to intraovarian factors during early follicular development remains to be elucidated. Because defective WT1 genes in mice result in early lethality, future studies using conditional cell-specific gene targeting may help clarify the role of WT1 in ovarian follicle development.
Recently, genes involved in early ovarian folliculogenesis such as c-kit [26] and growth/differentiation factor-9 (GDF-9) [27, 28] have been studied. The c-kit receptor tyrosine kinase present in the early growing oocyte stimulates oocyte growth after activation by its ligand, stem cell factor (SCF), expressed in granulosa cells [29]. Moreover, ovaries from mice carrying the SCF mutation are arrested at the one-layered cuboidal granulosa cell stage [26], implying an important role in the early stages of follicular development. Recently, GDF-9, a member of TGFß superfamily, has been shown to be present in oocytes at all stages of follicular development, except primordial follicles [27]. Ovaries from mice deficient in GDF-9 are arrested in the primary follicle stage, leading to infertility [28], suggesting that oocyte-derived GDF-9 could function during early follicle development. It is plausible that WT1 may play a role in early follicle development by coordinating the function of these early expressed growth factors in the suppression of ovarian differentiation genes.
The seminal event in early follicular development is the activation of nongrowing primordial follicles, a process that continues throughout reproductive life [1, 30]. The duration of the reproductive life span is determined by factors that influence the activation of follicles and the exhaustion of the pool of primordial follicles [31, 32]. Studies on factors regulating the rate of primordial follicle activation should take into consideration the possible suppression of early follicle development by the transcription suppressor WT1, and potential interactions between slow-growing early follicles and dormant primordial follicles.
In summary, the present data have suggested that a zinc-finger transcription factor, WT1, directly binds to inhibin- promoter and is highly expressed only in preantral follicles. The pattern of WT1 mRNA expression in granulosa cells is restricted to immature follicles over a range of avian and mammalian species and maintained throughout reproductive life. On the basis of these findings, we propose that WT1 serves as an important marker for the early stages of follicular development in different species, and may function as a suppressor of factors that stimulate granulosa cell proliferation and differentiation in immature follicles.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 650 725 7102; aaron.hsueh{at}stanford.edu
3 Current address: Hormone Research Center, Dept. of Biology, College of Natural Sciences, Chonnam National University, Kwangju, 500–757, Republic of Korea.
4 Current address: Department of Biology&Microbiology, California State University, Los Angeles, CA 90032.
5 Current address: INSERM U-407, Faculte de Medecine, Lyon-Sud, BP 12, 69 921 Oullins Cedex, France.
Accepted: September 18, 1998.
Received: August 19, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gene promoter. Mol Endocrinol 1995; 9:1356–1366. analogue, cloprostenol. Aust J Biol Sci 1980; 33:63–69.[Medline]This article has been cited by other articles:
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  J. Klattig, R. Sierig, D. Kruspe, B. Besenbeck, and C. Englert Wilms' Tumor Protein Wt1 Is an Activator of the Anti-Mullerian Hormone Receptor Gene Amhr2 Mol. Cell. Biol., June 15, 2007; 27(12): 4355 - 4364. [Abstract] [Full Text] [PDF]
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 K. L. Britt, P. K. Saunders, S. J. McPherson, M. L. Misso, E. R. Simpson, and J. K. Findlay Estrogen Actions on Follicle Formation and Early Follicle Development Biol Reprod, November 1, 2004; 71(5): 1712 - 1723. [Abstract] [Full Text] [PDF]
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R. B. Billiar, N. C. Zachos, M. G. Burch, E. D. Albrecht, and G. J. Pepe Up-Regulation of {alpha}-Inhibin Expression in the Fetal Ovary of Estrogen-Suppressed Baboons Is Associated with Impaired Fetal Ovarian Folliculogenesis Biol Reprod, June 1, 2003; 68(6): 1989 - 1996. [Abstract] [Full Text] [PDF]
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K. A. Logan, K. P. McNatty, and J. L. Juengel Expression of Wilms' Tumor Gene and Protein Localization During Ovarian Formation and Follicular Development in Sheep Biol Reprod, February 1, 2003; 68(2): 635 - 643. [Abstract] [Full Text] [PDF]
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H. Kim, S. You, J. Farris, L. K. Foster, Y.-J. Choi, and D. N. Foster Gonad-Specific Expression of Two Novel Chicken Complementary DNA Isoforms Biol Reprod, May 1, 2001; 64(5): 1473 - 1480. [Abstract] [Full Text]
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 A. Makrigiannakis, K. Amin, G. Coukos, J. L. Tilly, and C. Coutifaris Regulated Expression and Potential Roles of p53 and Wilms' Tumor Suppressor Gene (WT1) during Follicular Development in the Human Ovary J. Clin. Endocrinol. Metab., January 1, 2000; 85(1): 449 - 459. [Abstract] [Full Text]
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