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INTRACELLULAR SIGNAL SYSTEMS |
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: JoAnne S. Richards, Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: . joanner{at}bcm.tmc.edu
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Abstract |
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Introduction |
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Other factors that have been shown to impact ovarian cell function and follicular organization are members of the Wnt and fibroblast growth factor (FGF) families of regulatory peptides as well as members of the steroid nuclear receptor superfamily. Wnt-4, FGF-9, and steroidogenic factor-1 (SF-1) play key roles in early development of the ovary. Female mice null for Wnt-4 have sex-reversed ovaries that at birth are depleted of oocytes and contain supporting cells expressing genes characteristic of testis development such as MIS (8). Because of this severe ovarian phenotype and because these mice die shortly after birth owing to kidney defects, the expression and function of Wnt-4 in the adult ovary is not known. Which of the Frizzled (Fz) receptor(s) is activated by Wnt-4 during ovarian organogenesis is also not known. Mice null for FGF-9 exhibit male-to-female sex reversal of the reproductive system including the gonad and altered expression of sry-related, high mobility group box binding protein-9 and MIS (9). Mice null for SF-1 are born without gonads or adrenal glands and lack pituitary gonadotropes (10, 11). Results of recent studies indicate further that Wnt-4 in addition to SF-1 regulate expression of DAX-1 (dosage-sensitive sex-reversal, adrenal hypoplasia congenita critical region on the X chromosome, gene 1) (12), a putative repressor of SF-1 in the gonad. In the adult gonad, SF-1 has been shown to regulate the expression of genes such as aromatase (13), P450 side chain cleavage (14), MIS (15, 16), inhibin-
(17), and FSH receptor (18, 19). Therefore, Wnt-4 and possibly other members of the Wnt family may play critical roles in the function of the adult ovary via interactions with members of the TGF-ß, FGF, or steroid receptor superfamily. Of note, Wnt-4 as well as members of the TGF-ß and FGF families have also been shown to exert important functions in other reproductive organs such as the pituitary (20) and mammary glands (21, 22, 23, 24, 25).
Wnts are secreted extracellular signaling molecules that act locally to control diverse developmental processes such as cell fate specification, cell proliferation, and cell differentiation (26, 27). Wnt molecules transduce their signals by binding to serpentine G protein-coupled receptors of the Fz family to activate distinct signaling cascades (28, 29, 30). In the canonical Wnt/wg pathway, hyperphosphorylation of dishevelled (Dvl), a cytoplasmic scaffolding protein downstream of the Fz receptor, leads to subsequent inactivation of glycogen synthase kinase 3ß and the accumulation of ß-catenin. Soluble ß-catenin heterodimerizes with members of the T-cell factor/lymphoid enhancer factor (Tcf/Lef) family of transcription factors to regulate expression of selected target genes such as c-myc (31). Alternatively, Wnts can activate Fz receptors that signal via intracellular calcium, PKC, and/or calmodulin-dependent kinases (32). The Wnt/Fz signaling pathways are further modulated by coreceptors, proteoglycans, and/or arrow/low density lipoprotein receptor-related protein-5/-6 (33, 34, 35, 36, 37, 38, 39) and by antagonists such as the secreted Fz-related proteins (40). Thus, Wnt/Fz cellular signaling pathways are diverse and provide a potentially important local regulatory system by which ovarian cells may be critically dependent. Unfortunately, very little is known about the expression or function of this signaling system in the adult ovary.
On the basis of these considerations, this study was undertaken to determine whether specific Wnt and Fz genes were expressed in the adult ovary, their expression was hormonally regulated, and expression was restricted to specific cell types or stages of follicular development. For these studies we used both immature mouse and rat models in which ovarian follicular development, ovulation, and luteinization were regulated by exogenous administration of steroid and pituitary hormones. RT-PCR and in situ hybridization analyses were employed to survey the expression patterns of numerous Wnt and Fz transcripts in ovarian cells. Our results show that of the many Wnt and Fz, transcripts expressed in the ovary, Wnt-4, Fz-4, and Fz-1, appear to be abundantly expressed at specific stages of follicular growth and luteinization.
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Materials and Methods |
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Female 26-d-old Holtzman Sprague Dawley rats (Harlan Sprague Dawley, Inc.) were hypophysectomized (H). Untreated (H) 28-d-old rats were injected sc with 1.5 mg/0.2 ml 17ß-E2 (Sigma, St. Louis, MO) once daily for 3 d (HE). HE rats were then given sc injections of 1.0 µg/0.1 ml ovine FSH (NIH oFSH-16, National Hormone and Pituitary Agency, Rockville, MD) twice daily for 2 d (HEF). After the E2 and FSH treatments, the HEF rats were given a single ip injection of 10 IU hCG (Organon Special Chemicals) (HEF hCG). Similar to the mouse model, ovulation occurs in rats approximately 12–14 h after hCG.
PR-knockout (PRKO) mice were used in selected experiments because follicles develop normally in response to PMSG but fail to ovulate in response to hCG (41). PRKO female mice were injected with PMSG and hCG as described above and ovaries were isolated for RNA extractions. All animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals as approved by the Animal Care and Use Committee at Baylor College of Medicine (Houston, TX).
RNA isolation and RT-PCR
Total RNA was isolated from whole ovaries of immature (d 23) mice and PMSG- and hCG-treated mice using TRIzol Reagent (Life Technologies, Inc., Grand Island, NY) and purified as specified by the manufacturer. Each RNA sample was prepared from ovaries pooled from two to three animals. RNA was also extracted from whole ovaries of individual d-15-pregnant and postpartum mice, from ovaries of several neonatal mice at each of 2, 5, and 15 d of age, and from ovaries of individual hormone-primed PR heterozygote and knockout mice. Total RNA was similarly isolated from granulosa cells isolated from H, HE, HEF, and HEF hCG 12-h rat ovaries using TRIzol Reagent, from whole ovaries of individual rats at the HEF hCG 24-h time point and whole ovaries of several intact immature (d 26) Holtzman Sprague Dawley rats.
To survey the expression of Wnt signaling components in the ovary, total RNA (300 ng for Wnt-4; 500 ng for all other Wnts and Fzs) was reverse transcribed using poly-dT (Amersham Pharmacia Biotech, Newark, NJ; Roche Molecular/Boehringer, Chicago, IL) and avian myeloblastosis virus-reverse transcriptase (Promega Corp., Madison, WI) at 42 C for 75 min and 95 C for 5 min. Each reaction mixture was separated to two aliquots. A specific primer pair (see Table 1
) for each Wnt and Fz (Wnt-2, Wnt-3a, Wnt-4, Wnt-5a, Wnt-7a, Wnt-7b, Wnt-8, Wnt-10b, Wnt-11, Fz-1, Fz2, Fz-3, Fz-4, Fz-6, Fz-7, and Fz-8) was added to one aliquot, and primers for the ribosomal protein L-19 (42, 43) were added to the other aliquot as an internal control. Downstream components of the Wnt-Fz signaling pathway, Dvl-1 and Lef-1, were similarly amplified by RT-PCR. [32P]dCTP (ICN, Los Angeles, CA), Taq Polymerase and Thermocycle buffer (Promega Corp.) were also added to the reaction mixtures, and the reactions completed 30 cycles of PCR at 94 C for 1 min, 60 C for 2 min, and 72 C for 3 min. To determine the linear range of amplification for specific mRNAs, 300 ng RNA was reverse transcribed and amplified in a range of cycle numbers. Next, increasing amounts of RNA (75–1200 ng) were reverse transcribed and PCR amplified at a selected cycle number. RT-PCR (from 300 ng RNA) for Fz-1 and Fz-4 were repeated at 26 cycles (r = 0.974 and 0.911, respectively, after 25 cycles), 25 cycles for Wnt-4 (r = 0.989), and 20 cycles for L-19 (r = 0.975). Dvl-1 and Lef-1 were also amplified after 26 cycles. The resulting expression patterns for these signaling molecules were not different from those observed after 30 PCR cycles. A disintegrin and metalloproteinase with thrombospondin-like motifs (ADAMTS-1) was reverse transcribed and amplified after 20 PCR cycles (43). The amplified cDNA products were resolved on a 5% polyacrylamide gel, which was dried and exposed to film. The radioactive PCR product bands were quantified by using a Storm 860 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). Sequences for the PCR products were verified by sequencing.
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Cell extracts and Western blot analysis
Protein extracts were prepared from whole ovaries and isolated granulosa and residual cells of PMSG- and hCG-treated mice by homogenization in whole cell extract buffer (10 mM Tris, pH 7.4, 1 mM Na2EDTA, 1 mM dithiothreitol, 400 mM KCl, 10% glycerol, 5 µg/ml protease inhibitor cocktail I, 5 µg/ml protease inhibitor cocktail II, 1 mM phenylmethylsulfonyl fluoride, and 1 mM vanadate) and centrifugation at 4 C. Granulosa and residual cell extracts were further incubated with concanavalin A-Sepharose 4B beads (Sigma) to deplete extracts of cadherin-associated ß-catenin. Fifty micrograms whole ovary protein extracts or 20 µg granulosa or residual cell extracts were run on SDS-PAGE and transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA) by electrophoresis. Membranes were blocked with 5% Carnation milk (Nestle, Solon, OH) at room temperature for 1 h, followed by incubation with the monoclonal ß-catenin antibody (Transduction Laboratories, Inc., Lexington, KY) diluted 1:500 in 5% milk for another hour at room temperature. Blots were washed extensively in Tris-buffered saline containing 0.5% Tween-20, and then incubated for 1 h at room temperature with antimouse IgG peroxidase-linked antibody (Amersham Pharmacia Biotech) diluted 1:20,000 in 5% milk. After washing blots in Tris-buffered saline containing 0.5% Tween-20, enhanced chemiluminescence was performed using SuperSignal chemiluminescent detection reagents (Pierce Chemical Co., Rockford, IL), and immunoreactive proteins were visualized by autoradiography. DAX-1 protein was similarly detected using 5 µg granulosa cell and residual cell extracts of immature mouse and rat ovaries, 5 µg corpora luteal cell and residual cell extracts of d-15-pregnant mouse and rat ovaries, and a 1:500 dilution of the rabbit polyclonal DAX-1 antibody (K-17, Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Immunolocalization of DAX-1
HEF and d-14-pregnant rat ovaries were fixed, embedded, and sectioned as described above. Sections were deparaffinized in xylenes, quenched with 3% H2O2 in methanol for 10 min, and rehydrated. Sections were then incubated in 10 mM sodium citrate (pH 6.0) at 90 C for 20 min, cooled at room temperature for 20 min, and blocked with 20% goat serum in PBS for 1 h at room temperature. After blocking, rabbit polyclonal DAX-1 antibody (K-17, Santa Cruz Biotechnology, Inc.) diluted 1:100 in 10% goat serum was applied to the sections that were incubated at 4 C overnight. The next day, sections were washed in PBS and incubated for 1 h at room temperature with biotinylated antirabbit IgG (Vector Laboratories, Burlingame, CA) diluted 1:450 in 10% goat serum). As a control, some sections were incubated with only the secondary antibody. After washing with PBS, sections were incubated for 30 min with streptavidin-conjugated peroxidase diluted 1:500 in PBS. Sections were washed again in PBS, and localization of the primary antibody was visualized with diaminobenzidine (Vector Laboratories, Inc.), which yields a brown stain. Sections were dehydrated, cleared in xylenes, and mounted with Permount (Fisher Scientific).
Statistical analysis
The data were represented as the arithmetic mean plus or minus SEM. The t test was performed to analyze the significance of the difference between the two groups being compared. P < 0.05 was considered significant.
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Results |
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Fz-1 is expressed in granulosa cells of ovulatory follicles
RT-PCR analyses of Fz-1 revealed that Fz-1 mRNA increased in PMSG-primed mouse ovaries stimulated with hCG for 4–12 h (Fig. 1A, left). By 16 h after hCG, Fz-1 levels were markedly reduced and remained low in 48-h hCG-stimulated ovaries and d-15-pregnant mouse ovaries that contained functional corpora lutea as well as in postpartum mouse ovaries that contained regressing and newly formed corpora lutea. Specific induction of Fz-1 was also observed in granulosa cells isolated from hormone-primed rats after 12 h hCG, confirming the mouse data (Fig. 1A, right).
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), a time immediately preceding ovulation. Fz-1 was not detected by this method in corpora lutea (Fig. 1B).
Wnt-4 expression is selectively elevated in small follicles and in corpora lutea
Because Wnt-4 performs critical functions during early ovarian development, we analyzed Wnt-4 expression and localization in the adult rodent ovary. Wnt-4 mRNA was expressed in PMSG-treated mouse ovaries and increased in response to hCG (Fig. 2A, left). Wnt-4 was also expressed in hormone-primed rat ovaries and isolated rat granulosa cells, with elevated levels observed in granulosa cells after 12 h hCG and in whole rat ovaries after 24 h hCG (Fig. 2A, right). In situ hybridization analyses of Wnt-4 revealed it to be highly and selectively expressed in small growing follicles of ovaries from immature (d 23) mice (Fig. 2, B and C) as well as in small follicles of ovaries from hormone-stimulated and d-15-pregnant animals (Fig. 2B). Wnt-4 expression levels were also elevated in the corpora lutea of PMSG-primed mice after 24 h hCG and in the corpora lutea of d-15-pregnant mouse ovaries (Fig. 2B). Wnt-4 mRNA was also detected in large preovulatory and ovulatory follicles of ovaries from mice treated with PMSG or PMSG plus hCG 12 h (Fig. 2B).
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). In situ hybridization analyses were performed using ovaries collected from newborn (d 0), 2-, 5-, and 15-d-old mice. In d-0, d-2, and d-5 mouse ovaries, Wnt-4 mRNA was not easily detected above background levels (Fig. 3B). At d 0, follicles have not yet formed. Rather, the ovaries contain nests of oocytes that are arrested in meiosis prophase I. Primordial follicles, which consist of a primary oocyte surrounded by a single layer of flattened granulosa cells, begin to form by d 2 and are more obvious in ovaries of 5-d-old mice. By d 15, numerous growing primary follicles containing one or a few layers of cuboidal granulosa cells are evident, and in these follicles Wnt-4 mRNA is clearly detected (Fig. 3B).
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, left). Similarly, Fz-4 was expressed in hormonally primed rat ovaries (Fig. 4A, right). In situ hybridization analyses of Fz-4 revealed increased expression in corpora lutea of hormone-stimulated mice as well as in the corpora lutea of a d-15-pregnant mouse ovary (Fig. 4B). The expression of both Wnt-4 and Fz-4 in corpora lutea suggests that Wnt-4 may be a ligand for Fz-4 in these structures and that Wnt4/Fz-4 signaling may be important for regulation of corpora luteal cell functions. Unlike Wnt-4, Fz-4 expression was not visualized in small follicles (Fig. 4, B and C) despite RT-PCR data indicating the presence of Fz-4 mRNA. As shown above, low levels of Fz-4 and Fz-1 were also detected in neonatal mouse ovaries by RT-PCR (Fig. 3A).
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), expression of downstream components of the Wnt/ß-catenin pathway, Dvl-1 and Lef-1, was analyzed. Our results showed that mRNAs encoding the scaffold protein Dvl-1 (Fig. 5A) and the transcription factor Lef-1 (Fig. 5B) are expressed in the mouse ovary but do not appear to be regulated by hormones. In addition, ß-catenin protein was detected in whole mouse ovaries as well as in soluble (cadherin-free) cytosolic extracts of isolated granulosa cells and residual cells by Western blot analyses (Fig. 5C). Together, these results indicate that the Wnt/ß-catenin pathway has the potential to be functional in the mouse ovary, especially in connection with Fz-1.
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). However, after 12 h of hCG stimulation, Fz-1 mRNA was lower (P < 0.05) in PRKO mouse ovaries, compared with the PR heterozygote (Fig. 6A).
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). Not unexpectedly, therefore, we observed that Wnt-4 mRNA was induced by hCG equally well in hormonally primed PR heterozygote and knockout mouse ovaries (Fig. 6B).
In previous studies, ADAMTS-1 has been shown to be induced by hCG in ovaries of normal mice but not in ovaries of PRKO mice that fail to ovulate (43). Therefore, as a control for the quality of the RNA and the effects of hCG and PR on gene expression in these same samples, ADAMTS-1 mRNA was analyzed. As shown (Fig. 6C), ADAMTS-1 mRNA was increased 10- to 40-fold by hCG in ovaries of heterozygous mice. However, in the PRKO mouse ovaries, ADAMTS-1 mRNA was increased 3- to 4-fold by hCG, remaining 50–80% lower than that observed in the heterozygous ovaries 8–12 h after hormone treatment (Fig. 6C).
DAX-1 is expressed by multiple cell types in the rodent ovary
Because recent studies implicate DAX-1 as a target of Wnt-4 signaling (12, 49), immunohistochemistry and Western blot analyses were performed to localize and quantitate DAX-1 in the rodent ovary. As shown in Fig. 7A, DAX-1 is expressed by many cell types including thecal and interstitial cells, granulosa cells, and corpora lutea cells, in which nuclear staining for DAX-1 is clearly evident. No staining was observed in the presence of secondary antibody alone (data not shown). Western blot analysis confirmed the specificity of the DAX-1 antibody and expression of DAX-1 in granulosa cells of small follicles, preovulatory follicles, and corpora luteal cells (Fig. 7B).
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Discussion |
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Of the Fz receptors surveyed by RT-PCR in the rodent ovary, Fz-1 was selected for further analyses because it was up-regulated 4–12 h in response to hCG and then decreased as follicles luteinized. In the mouse ovary, Fz-1 expression was first up-regulated in thecal and interstitial cells and then specifically in granulosa cells of large ovulatory follicles. The selective and transient induction of Fz-1 in granulosa cells by LH suggested that the Fz-1 gene was transcriptionally activated by factors known to impact ovulation. One of these is PR, a gene that is also induced by LH and is obligatory for ovulation because mice null for PR fail to ovulate even when stimulated with exogenous hormones (41, 43, 47). Analyses of Fz-1 expression in heterozygous and PRKO mouse ovaries indicate that LH induces the initial increase in Fz-1 message independently of PR. However, by 12 h after hCG, a time just preceding ovulation, Fz-1 mRNA is reduced in the ovaries of the PRKO mice, compared with that of the heterozygous mice. These results show that the pattern of Fz-1 expression is altered in the ovaries of PRKO mice but not as drastically as that of ADAMTS-1, another LH- and PR-regulated gene. The observed smaller decrease in Fz-1 mRNA, compared with that of ADAMTS-1 is likely because of the fact that Fz-1 is expressed by theca and granulosa cells, which both contribute to the RT-PCR products generated from whole ovarian RNA, and ADAMTS-1 expression is restricted to granulosa cells, the site of PR regulation. Nevertheless, the high levels of Fz-1 in granulosa cells of large ovulatory follicles suggest that signaling through the Fz-1 receptor may regulate genes that coordinate with PR-induced genes to control ovulation. Recently, rat Fz-1 has been shown to activate the ß-catenin pathway via G protein signaling (30). Therefore, in the ovulating follicle, Fz-1 may control granulosa cell movement or differentiation or regulate genes that impact follicle rupture or luteinization.
The expression patterns of Wnt-4 and Fz-4 transcripts in the adult ovary are clearly distinct from that of Fz-1. Fz-4 is expressed in the adult rodent ovary with specific localization and increased detection by in situ hybridization in corpora lutea of PMSG-hCG-treated mice and pregnant mice. Likewise, Wnt-4 showed specific localization to corpora lutea of hormonally primed and pregnant mice. That Wnt-4 and Fz-4 are coexpressed in corpora lutea suggests that Fz-4 may function as a receptor for Wnt-4 in this tissue. Although the exact functional role(s) of Wnt-4 and Fz-4 in this tissue is not known, it is tempting to speculate that they impact maintenance of this terminally differentiated tissue.
Evidence that Wnt-4 plays a key role in earlier stages of ovarian function is more conclusive. First, mice null for Wnt-4 exhibit female-to-male sex reversal of the gonads (8). At birth, these ovaries lack oocytes and exhibit an abnormal male-like pattern of steroidogenic activity and expression of MIS. These studies indicated that Wnt-4 might be important for postmeiotic maintenance of oocytes (8). This hypothesis is supported by the data presented herein that show that Wnt-4 is expressed in ovaries of neonatal mice between 0 and 5 d old when primordial and primary follicles are being formed. Wnt-4 is also expressed in small growing follicles of ovaries of mice 15 d old and adult mice, indicating that this ligand impacts an Fz signaling cascade that controls the initiation of follicular growth. Although the specific role(s) of Wnt-4 in these early stages of follicle formation and growth is not known, some hypotheses can be presented. In the embryonic gonad, the pattern of Wnt-4 expression is similar to that of DAX-1 (8, 53). More recently, overexpression of Wnt-4 in Sertoli cells and Leydig cells up-regulates expression of DAX-1 (12), indicating that one function of Wnt-4 in the ovary may also be to regulate DAX-1. This observation is supported by the ability of ß-catenin to enhance SF-1-stimulated transactivation of the DAX-1 promoter via Tcf/Lef promoter elements (49). As a corepressor of the orphan nuclear receptor SF-1, ovarian DAX-1 may antagonize the transcriptional activation of genes that are regulated by SF-1 such as aromatase (13), P450 side-chain cleavage (14), 17-hydroxylase (54), FSH receptor (18, 19), MIS (15, 16), and inhibin- (17). The expression of these genes is low in small growing follicles of immature mice and rats (1, 55). Thus, it is tempting to speculate that a Wnt-4/Fz pathway present in small follicles controls certain genes, like DAX-1, in the adult ovary by regulating the functional activity of SF-1.
However, the role of Wnt-4 in the ovary appears to be much more complex. DAX-1 remains expressed (albeit at a lower level) in mice null for Wnt-4 (8, 12), likely because of the potent control of DAX-1 expression by SF-1. However, only some but not all SF-1 regulated genes are elevated in the Wnt-4-null mice (8). In addition, mice null for DAX-1 appear to have normal ovarian function (56). Thus, although the expression of Wnt-4 in small follicles may suppress steroidogenesis at this stage of development, a critical role for DAX-1 is not entirely clear. Even more striking, Wnt-4 and Fz-4 are elevated in luteal cells that are highly steroidogenic and contain nuclear Dax-1 protein. Therefore, it is tempting to speculate that the Wnt-4/Fz pathway that operates in small follicles is different from the Wnt-4/Fz-4 pathways that appear, but has not yet been proven, to be dominant in luteal cells. In addition, the specific downstream effectors of Wnt/Fz signaling may change as follicles terminally differentiate to luteal cells, thereby controlling distinct patterns of gene expression. Recent studies have shown that luteal cells exhibit elevated expression of specific kinases including Sgk (57, 58) and an MAPK pathway (59). Whether these kinases are targets (or mediators) of Wnt/Fz signaling is not known.
Based on the effects of Wnt/Fz signaling and interactions with BMP/TGF-ß and FGF signaling pathways in other tissues such as the developing mammary gland (21, 22, 23, 24, 25), pituitary gland (20), kidney (60), heart (61), and adipose (62), it is likely that Wnts are controlling the activity of multiple cellular signaling pathways in the ovary. However, unlike the mammary gland, Wnt-4 is not always coexpressed with PR in the ovary and does not appear to be a target of PR in ovulating follicles. Unlike the kidney, ovarian Wnt-4 is not regulating epithelial cell transformation of mesenchymal cells. Rather, in the developing ovary, Wnt-4 appears to dictate ovarian vs. testis morphogenic and genetic programs. In the indifferent gonad, Wnt-4 is expressed at embryonic d 11.5 but is not present in the developing testis (8). In contrast, FGF-9 is not expressed in the ovary, and mice null for FGF-9 exhibit male-to-female sex reversal (9). In the absence of FGF-9, the expression of genes involved in testis development, namely sry-related, high mobility group binding protein-9 and MIS, are reduced or lacking. Because mice null for Wnt-4 exhibit female-to-male sex reversal of the gonads with elevated expression of MIS, these Wnt-4 and FGF-9 pathways appear to be highly antagonistic in gonadogenesis. In the adult ovary, it is also possible that products of Wnt-4 signaling in granulosa cells antagonize (or in some cases synergize) with the actions of FGF or BMP molecules that are expressed in theca cells or oocytes of growing follicles.
The localization and regulation of Wnt-4, Fz-4, and Fz-1 in the adult rodent ovary, combined with the evidence for critical roles for Wnt-4 and FGF-9 in ovary and testis development, respectively, indicate that Wnt/Fz signaling is important for the growth and development of ovarian follicles. The identification of these ovarian-derived regulatory molecules provides new insight into the complex systems that impact ovarian cell function. When more information becomes available about which members of the BMP and FGF families are also expressed in the ovary and what specific functions Wnt signaling pathways control, a new intraovarian regulatory network may be clearly defined.
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Footnotes |
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Abbreviations: ADAMTS-1, A disintegrin and metalloproteinase with thrombospondin-like motifs; BMP, bone morphogenic factor; DAX-1, dosage-sensitive sex-reversal, adrenal hypoplasia congenita critical region on the X chromosome, gene 1; Dvl, dishevelled; FGF, fibroblast growth factor; Fz, Frizzled; H, hypophysectomized; hCG, human CG/Pregnyl; HE, hypophysectomized with 17ß-E2; HEF, hypophysectomized with 17ß-E2 and ovine FSH; HEF hCG, HEF rats given hCG; MIS, Müllerian-inhibiting substance; PRKO, PR knockout; SF-1, steroidogenic factor-1; Tcf/Lef, T-cell factor/lymphoid enhancer factor.
Received August 27, 2001.
Accepted for publication November 7, 2001.
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References |
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-promoter by steroidogenic factor-1 and cyclic adenosine 3',5'-monophosphate. Mol Endocrinol 14:66–81o and Gt. Proc Natl Acad Sci USA 96:14383–14388-hydroxylase/c17–20 lyase). Mol Endocrinol 10:147–158This article has been cited by other articles:
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 C. Ottolenghi, E. Pelosi, J. Tran, M. Colombino, E. Douglass, T. Nedorezov, A. Cao, A. Forabosco, and D. Schlessinger Loss of Wnt4 and Foxl2 leads to female-to-male sex reversal extending to germ cells Hum. Mol. Genet., December 1, 2007; 16(23): 2795 - 2804. [Abstract] [Full Text] [PDF]
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C. Stocco, C. Telleria, and G. Gibori The Molecular Control of Corpus Luteum Formation, Function, and Regression Endocr. Rev., February 1, 2007; 28(1): 117 - 149. [Abstract] [Full Text] [PDF]
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M. Shimada, I. Hernandez-Gonzalez, I. Gonzalez-Robanya, and J. S. Richards Induced Expression of Pattern Recognition Receptors in Cumulus Oocyte Complexes: Novel Evidence for Innate Immune-Like Functions during Ovulation Mol. Endocrinol., December 1, 2006; 20(12): 3228 - 3239. [Abstract] [Full Text] [PDF]
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 T. N. Parakh, J. A. Hernandez, J. C. Grammer, J. Weck, M. Hunzicker-Dunn, A. J. Zeleznik, and J. H. Nilson Follicle-stimulating hormone/cAMP regulation of aromatase gene expression requires beta-catenin PNAS, August 15, 2006; 103(33): 12435 - 12440. [Abstract] [Full Text] [PDF]
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I. Hernandez-Gonzalez, I. Gonzalez-Robayna, M. Shimada, C. M. Wayne, S. A. Ochsner, L. White, and J. S. Richards Gene Expression Profiles of Cumulus Cell Oocyte Complexes during Ovulation Reveal Cumulus Cells Express Neuronal and Immune-Related Genes: Does this Expand Their Role in the Ovulation Process? Mol. Endocrinol., June 1, 2006; 20(6): 1300 - 1321. [Abstract] [Full Text] [PDF]
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 D. Boerboom, L. D. White, S. Dalle, J. Courty, and J. S. Richards Dominant-Stable {beta}-Catenin Expression Causes Cell Fate Alterations and Wnt Signaling Antagonist Expression in a Murine Granulosa Cell Tumor Model Cancer Res., February 15, 2006; 66(4): 1964 - 1973. [Abstract] [Full Text] [PDF]
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V. Sriraman, M. D. Rudd, S. M. Lohmann, S. M. Mulders, and J. S. Richards Cyclic Guanosine 5'-Monophosphate-Dependent Protein Kinase II Is Induced by Luteinizing Hormone and Progesterone Receptor-Dependent Mechanisms in Granulosa Cells and Cumulus Oocyte Complexes of Ovulating Follicles Mol. Endocrinol., February 1, 2006; 20(2): 348 - 361. [Abstract] [Full Text] [PDF]
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M. Hsieh, D. Boerboom, M. Shimada, Y. Lo, A. F. Parlow, U. F.O. Luhmann, W. Berger, and J. S. Richards Mice Null for Frizzled4 (Fzd4-/-) Are Infertile and Exhibit Impaired Corpora Lutea Formation and Function Biol Reprod, December 1, 2005; 73(6): 1135 - 1146. [Abstract] [Full Text] [PDF]
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D. Boerboom, M. Paquet, M. Hsieh, J. Liu, S. P. Jamin, R. R. Behringer, J. Sirois, M. M. Taketo, and J. S. Richards Misregulated Wnt/{beta}-Catenin Signaling Leads to Ovarian Granulosa Cell Tumor Development Cancer Res., October 15, 2005; 65(20): 9206 - 9215. [Abstract] [Full Text] [PDF]
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 M. Heikkila, R. Prunskaite, F. Naillat, P. Itaranta, J. Vuoristo, J. Leppaluoto, H. Peltoketo, and S. Vainio The Partial Female to Male Sex Reversal in Wnt-4-Deficient Females Involves Induced Expression of Testosterone Biosynthetic Genes and Testosterone Production, and Depends on Androgen Action Endocrinology, September 1, 2005; 146(9): 4016 - 4023. [Abstract] [Full Text] [PDF]
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C. M. Bastida, A. Cremades, M. T. Castells, A. J. Lopez-Contreras, C. Lopez-Garcia, F. Tejada, and R. Penafiel Influence of Ovarian Ornithine Decarboxylase in Folliculogenesis and Luteinization Endocrinology, February 1, 2005; 146(2): 666 - 674. [Abstract] [Full Text] [PDF]
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E. Jansen, J. S. E. Laven, H. B. R. Dommerholt, J. Polman, C. van Rijt, C. van den Hurk, J. Westland, S. Mosselman, and B. C. J. M. Fauser Abnormal Gene Expression Profiles in Human Ovaries from Polycystic Ovary Syndrome Patients Mol. Endocrinol., December 1, 2004; 18(12): 3050 - 3063. [Abstract] [Full Text] [PDF]
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 D. L. Russell, K. M. H. Doyle, S. A. Ochsner, J. D. Sandy, and J. S. Richards Processing and Localization of ADAMTS-1 and Proteolytic Cleavage of Versican during Cumulus Matrix Expansion and Ovulation J. Biol. Chem., October 24, 2003; 278(43): 42330 - 42339. [Abstract] [Full Text] [PDF]
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M. Hsieh, S. M. Mulders, R. R. Friis, A. Dharmarajan, and J. S. Richards Expression and Localization of Secreted Frizzled-Related Protein-4 in the Rodent Ovary: Evidence for Selective Up-Regulation in Luteinized Granulosa Cells Endocrinology, October 1, 2003; 144(10): 4597 - 4606. [Abstract] [Full Text] [PDF]
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J. R. Wood, V. L. Nelson, C. Ho, E. Jansen, C. Y. Wang, M. Urbanek, J. M. McAllister, S. Mosselman, and J. F. Strauss III The Molecular Phenotype of Polycystic Ovary Syndrome (PCOS) Theca Cells and New Candidate PCOS Genes Defined by Microarray Analysis J. Biol. Chem., July 11, 2003; 278(29): 26380 - 26390. [Abstract] [Full Text] [PDF]
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 K. Surendran and T. C. Simon CNP gene expression is activated by Wnt signaling and correlates with Wnt4 expression during renal injury Am J Physiol Renal Physiol, April 1, 2003; 284(4): F653 - F662. [Abstract] [Full Text] [PDF]
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G. E. Owens, R. A. Keri, and J. H. Nilson Ovulatory Surges of Human CG Prevent Hormone-Induced Granulosa Cell Tumor Formation Leading to the Identification of Tumor-Associated Changes in the Transcriptome Mol. Endocrinol., June 1, 2002; 16(6): 1230 - 1242. [Abstract] [Full Text] [PDF]
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J. S. Richards, S. C. Sharma, A. E. Falender, and Y. H. Lo Expression of FKHR, FKHRL1, and AFX Genes in the Rodent Ovary: Evidence for Regulation by IGF-I, Estrogen, and the Gonadotropins Mol. Endocrinol., March 1, 2002; 16(3): 580 - 599. [Abstract] [Full Text] [PDF]
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