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Endocrinology Vol. 143, No. 3 898-908
Copyright © 2002 by The Endocrine Society

INTRACELLULAR SIGNAL SYSTEMS

Regulated Expression of Wnts and Frizzleds at Specific Stages of Follicular Development in the Rodent Ovary

Minnie Hsieh, Mac A. Johnson, Norman M. Greenberg and JoAnne S. Richards

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Wnt ligands and Frizzled (Fz) G protein-coupled receptors impact cell fate, including embryonic development of the ovary. Because the role of these regulatory molecules during follicular development in the adult is not known, an RT-PCR survey was done. Wnt-4, Fz-4, and Fz-1 were among the transcripts detected, and each exhibited a specific pattern of expression. Fz-1 mRNA was low in preovulatory follicles of PMSG-treated mice but was increased within 4–12 h after an ovulatory surge of human CG. By in situ analysis, Fz-1 transcripts increased first in the theca cells and then in the granulosa cells of ovulating follicles but were low in corpora lutea. In contrast, Wnt-4, a critical factor in early ovarian development, was expressed in small preantral follicles. In addition, Wnt-4 was detected in preovulatory follicles and exhibited high levels in corpora lutea. A potential receptor for Wnt-4 in corpora lutea is Fz-4 that was also elevated in this tissue. Although Wnt-4 has been shown to function downstream of the PR in other tissues, Wnt-4 was not altered in follicles of PR-null mice that fail to ovulate. Rather expression of Fz-1 was lower in ovaries of PR knockout mice, compared with normal littermates. Thus, specific Wnt/Fz are expressed at distinct stages of follicular development, suggesting multiple functions for this signaling pathway in the ovary.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FOLLICULAR GROWTH, OVULATION, and luteinization are dynamic processes that depend on changes in cell-cell interactions, cell differentiation, and extracellular matrix remodeling. These events are controlled, in part, by the pituitary gonadotropins FSH and LH (1). In addition, these events are regulated by ovarian-derived factors. Members of the TGF-ß family such as growth differentiation factor-9, Müllerian-inhibiting substance (MIS), and bone morphogenic factors (BMP-4, -7, and -15) have all been shown to exert specific effects on ovarian cell function. The oocyte-derived factor growth differentiation factor-9 plays a key role in organizing the theca cell layer in small follicles (2), and another oocyte-specific factor BMP-15 appears to regulate cumulus expansion in preovulatory follicles (3). The granulosa cell-expressed factor MIS appears to promote the growth of preantral follicles while inhibiting follicle cell differentiation (4, 5, 6). The theca cell-derived factors BMP-4 and -7 have been shown to regulate granulosa cell responsiveness to FSH in vitro (7).

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-{alpha} (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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and hormone treatments
Immature 23-d-old C57BL/6 female mice (Harlan Sprague Dawley, Inc., Chicago, IL) were injected ip with 4 IU PMSG (Professional Compounding Center of America, Houston, TX) to stimulate follicular growth. After 48 h, the mice were injected ip with 5 IU human CG/Pregnyl (hCG; Organon Special Chemicals, West Orange, NJ), an LH-like molecule used to induce ovulation and luteinization. In this model, ovulation occurs approximately 12–14 h after hCG. Ovaries were isolated from these hormone-stimulated mice at selected time intervals and used for extraction of RNA and protein or fixed for in situ hybridization analyses. Ovaries were also isolated from neonatal mice at 0, 2, 5, and 15 d of age and from d-15-pregnant and d-1-postpartum mice for preparation of RNA or in situ analyses.

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 1Go) 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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  		Table 1. Sequences for PCR primers

 
In situ hybridization
In situ hybridization was performed as described by Wilkensen (44) and as previously shown in our laboratory (45). Briefly, the riboprobe in vitro transcription systems kit (Promega Corp.) was used to make [35S]UTP-labeled antisense and sense probes of mouse Fz-1, Fz-4, and Wnt-4 cDNAs. Each cDNA was generated by RT-PCR amplification, cloned into the EcoRV site of the pBluescript SK vector after T tailing, and verified by sequencing. Ovaries were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 7 µm onto Fisher-brand Superfrost Plus microscope slides (Fisher Scientific, Pittsburgh, PA). Tissue sections were rehydrated, treated with 20 µg/ml proteinase K and 0.1 M triethanolamine/acetic anhydride, dehydrated, and incubated with radiolabeled riboprobe overnight at 55 C. The next day, slides were washed at high stringency, dried, and exposed to X-OMAT film (Kodak, Rochester, NY) overnight to determine the specificity and intensity of the probe. Slides were dipped in photographic NTB-2 emulsion, exposed at 4 C for an appropriate length of time, developed with D-19 developer and fixer (Kodak), and stained with hematoxylin. Tissue histology was observed by light-field illumination, and dark-field illumination was used to visualize the mRNA probe. In situ analyses of Fz-1, Fz-4, and Wnt-4 were performed by preparing slides for each time point on which multiple sections of ovaries from each of three mice were arranged.

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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of Wnts and Fzs in the mouse ovary
To determine which Wnts and Fzs are expressed in the adult mouse ovary, an RT-PCR survey was performed using RNA isolated from mouse ovaries treated with PMSG to stimulate follicular growth and hCG-stimulated ovaries that ovulate and luteinize. Our results showed that multiple Wnt and Fz transcripts are expressed at varying levels in the ovary. These include Wnt-2, Wnt-3a, Wnt-4, Wnt-5a, Wnt-7a, Wnt-8, Wnt-10b, Wnt-11, Fz-1, Fz-2, Fz-3, Fz-4, Fz-6, and Fz-7 (data not shown). Wnt-7b and Fz-8, however, were not detected in the mouse ovary by this method. Based on the relevance to ovarian development (8) and the apparent hormonal regulation of specific RNAs, we have focused in this study primarily on Fz-1, Fz-4, and Wnt-4 as discussed below.

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. 1AGo, 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. 1AGo, right).



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  		Figure 1. Fz-1 is induced by LH in ovulating follicles of rodent ovaries. A, Left, Representative semiquantitative RT-PCR analysis with corresponding autoradiographs shows induction of Fz-1 mRNA in the mouse ovary between 4 and 12 h after hCG. In this and in subsequent figures (unless specified), RT-PCR was performed multiple times on at least two separate time courses of PMSG-hCG-treated mouse ovary RNAs (for each time course, each time point was prepared from ovaries of two to three animals) using 300 ng whole ovarian RNA. The relative signal was determined by quantifying radioactive PCR product bands as described in the methods and normalizing Fz-1 expression levels to that for L-19. Fz-1 and L-19 underwent 26 and 20 PCR cycles, respectively. Right, Fz-1 mRNA was also induced in hormonally primed rat granulosa cells 12 h after an ovulatory stimulus of hCG (GC, granulosa cells; W, whole ovaries; CL, corpora luteal cells). RT-PCR was performed on 500 ng RNA at 30 PCR cycles. B, In situ hybridization analyses of mouse ovaries shows an initial increase in Fz-1 in thecal and interstitial cells after 4 h hCG and then a shift in expression to granulosa cells of large ovulatory follicles between 8 and 12 h hCG. Images are shown at 10x magnification (5x for sense) in both dark (left panels) and light (right panels) field illumination (PO, preovulatory follicle; T, thecal cell layer; GC, granulosa cells; Ov, ovulatory follicle; CL, corpus luteum).

 
In situ hybridization analyses of mouse ovaries showed that the increase in Fz-1 expression occurred initially in theca and interstitial cells after 4 h hCG and then specifically in granulosa cells of large ovulatory follicles between 8 and 12 h after hCG (Fig. 1BGo), a time immediately preceding ovulation. Fz-1 was not detected by this method in corpora lutea (Fig. 1BGo).

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. 2AGo, 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. 2AGo, 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. 2Go, B and C) as well as in small follicles of ovaries from hormone-stimulated and d-15-pregnant animals (Fig. 2BGo). 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. 2BGo). 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. 2BGo).



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  		Figure 2. Expression of Wnt-4 is increased in small growing follicles and corpora lutea. A, Left, Representative semiquantitative RT-PCR analysis with corresponding autoradiographs shows an increase in Wnt-4 mRNA levels in mouse ovaries following hCG stimulation. RT-PCR was performed as described in Fig. 1Go but with 25 PCR cycles for Wnt-4. Right, Wnt-4 mRNA was induced in rat granulosa cells after 12 h hCG and in whole ovaries after 24 h hCG. RT-PCR was performed on 300 ng RNA at 25 PCR cycles. B, In situ hybridization analyses of Wnt-4 shows increased expression in small follicles and in corpora lutea. Images are shown at 5x magnification in dark (left panels) and light (right panels) field illumination (sf, small follicle; PO, preovulatory follicle; Ov, ovulatory follicle; CL, corpus luteum). Mouse ovaries treated with 48 h hCG also showed elevated levels of Wnt-4 in corpora lutea, and no staining above background levels was observed with the sense riboprobe in these same ovaries (data not shown). C, Immature (d 23) mouse ovary from (B) shown at higher magnification (10x). Arrows point to small Wnt-4-positive follicles containing a few layers of granulosa cells.

 
The expression of Wnt-4 in small primary follicles indicated that it might function to regulate earlier stages of follicle growth and organization. Therefore, additional analyses were performed on RNA of neonatal mouse ovaries. Results showed that Wnt-4, as well as Fz-1 and Fz-4, is expressed in ovaries of 2-, 5-, and 15-d-old mice (Fig. 3AGo). 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. 3BGo). 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. 3BGo).



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  		Figure 3. Wnt-4 is expressed in neonatal mouse ovaries. A, Wnt-4, Fz-4, and Fz-1 are expressed in neonatal mouse ovaries by RT-PCR. RT-PCR was performed on 300 ng RNA at 25 PCR cycles for Wnt-4 and 30 cycles for Fz-4 and Fz-1. B, In situ hybridization analyses show low expression of Wnt-4 by primordial follicles in d-0, d-2, and d-5 mouse ovaries. By d 15, specific staining for Wnt-4 mRNA is detected in some growing primary follicles (arrowheads). The dark and light field images in the upper two rows (20x magnification; 10x for the d-15 ovary) are shown at higher magnification (boxed areas at 40x magnification; 20x for the d-15 ovary) in the lower two rows. (o, oocyte; pmf, primordial follicle; pf, primary follicle).

 
Fz-4 is expressed in the corpus luteum
Fz-4, a potential receptor for Wnt-4, was expressed in hormone-stimulated, pregnant, and postpartum mouse ovaries (Fig. 4AGo, left). Similarly, Fz-4 was expressed in hormonally primed rat ovaries (Fig. 4AGo, 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. 4BGo). 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. 4Go, 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. 3AGo).



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  		Figure 4. Expression of Fz-4 is increased in corpora lutea. A, Left, Representative semiquantitative RT-PCR analysis with corresponding autoradiographs shows general expression of Fz-4 in hormone-primed mouse ovaries. Right, Similar to the mouse data, Fz-4 appears generally expressed in rat ovaries. RT-PCR analyses in both mouse and rat ovaries were performed as described in Fig. 1Go. B, In situ hybridization analyses of Fz-4 shows highest expression in corpora lutea and low staining in small follicles. Images are shown at 5x magnification in dark (left panels) and light (right panels) field illumination (sf, small follicle; PO, preovulatory follicle; Ov, ovulatory follicle; CL, corpus luteum). No staining above background levels was observed in 48 h hCG-treated mouse ovaries hybridized with the sense riboprobe (data not shown). C, Immature (d 23) mouse ovary from (B) shown at higher magnification (10x). Arrows point to small follicles containing a few layers of granulosa cells with low staining for Fz-4 mRNA.

 
Expression of downstream components of the Wnt/ß-catenin pathway
Because the Wnt/ß-catenin pathway is activated by selected Wnts and Fzs, including rat Fz-1 (30, 46) that is induced in response to hCG (Fig. 1Go), 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. 5AGo) and the transcription factor Lef-1 (Fig. 5BGo) 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. 5CGo). 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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  		Figure 5. Downstream components of the Wnt/ß-catenin pathway are present and not hormonally regulated in the mouse ovary. Representative semiquantitative RT-PCR analysis with corresponding autoradiographs shows expression of Dvl-1 (A) and Lef-1 (B) in hormone-stimulated mouse ovaries. RT-PCR analyses for Dvl-1 and Lef-1 were performed as described in Fig. 1Go. C, Western blot analyses of ß-catenin in protein extracts from whole mouse ovaries (WO) or from cytosolic extracts from isolated granulosa cells (GC) or residual cells (RC).

 
Expression of Fz-1 but not Wnt-4 is altered in ovaries of mice null for the PR
The PR is induced by the LH/hCG surge and has been shown to be required for ovulation because ovaries of PRKO animals failed to ovulate even when stimulated with PMSG and hCG (41, 43, 47). Because Fz-1 mRNA is specifically induced before and around the time of ovulation, we analyzed Fz-1 expression in ovaries of hormonally stimulated PR heterozygote and knockout mice to determine whether Fz-1 was PR regulated. The results indicate that Fz-1 is induced in both PR heterozygote and PRKO mouse ovaries 4–8 h after hCG (Fig. 6AGo). 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. 6AGo).



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  		Figure 6. PR impacts expression of Fz-1 and ADAMTS-1 but not Wnt-4. Semiquantitative RT-PCR analyses of Fz-1 (A), Wnt-4 (B), and ADAMTS-1 (C) using RNA from individual PMSG-hCG treated PR heterozygote (solid bars) and PRKO (open and hatched bars) mouse ovaries. Fz-1 was induced by hCG in both heterozygote and PRKO animals, although Fz-1 mRNA levels were reduced in two different PRKO mouse ovaries, compared with the heterozygote ovary after 12 h hCG. Wnt-4 was induced equally in both heterozygote and PRKO mouse ovaries following hCG stimulation. Expression of ADAMTS-1 mRNA, which is PR regulated (43 ), was more than 80% lower in PRKO ovaries, compared with heterozygote ovaries after 12 h hCG. RT-PCR analyses for each gene were repeated with additional PR heterozygote (n = 4) and PRKO (n = 5) mouse ovary samples at the PMSG-hCG 12-h time point. Error bars represent mean plus or minus SEM (*, P < 0.05; **, P < 0.001). RT-PCR reactions were performed as described in Fig. 1Go but with whole ovarian RNA from individual PR heterozygote and PRKO animals, 25 PCR cycles for Wnt-4, and 20 cycles for ADAMTS-1.

 
In the mouse mammary gland, Wnt-4 and PR colocalize in mammary epithelial cells (21). Wnt-4 was further shown to function downstream of progesterone signaling to mediate side branching of mammary ducts early in pregnancy (21). In the mouse ovary, however, Wnt-4 and PR mRNAs do not always colocalize because Wnt-4 but not PR (43, 48) is expressed in small follicles and corpora lutea (Fig. 2BGo). 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. 6BGo).

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. 6CGo), 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. 6CGo).

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. 7AGo, 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. 7BGo).



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  		Figure 7. DAX-1 is expressed by multiple cell types in the rodent ovary. A, Immunohistochemistry with an anti-DAX-1 antibody and diaminobenzidine detection was performed on whole ovarian sections from a hypophysectomized rat treated with E2 and FSH (left) and from a d-14-pregnant rat (right). DAX-1 protein was detected in theca and interstitial cells (T/I), granulosa cells (GC), and corpora luteal cells (CL), where nuclear staining is apparent (inset, higher magnification). B, Western blot analyses confirm the presence of DAX-1 in protein extracts prepared from granulosa and residual cells of immature rats and mice and in protein extracts of corpora lutea and residual cells of a d-15-pregnant rat and d-15-pregnant mouse.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Knockout studies of various Wnt signaling molecules led to the discovery that specific Wnts perform critical functions during early development of the ovary and other reproductive tissues (8, 50, 51, 52). In this study, we report for the first time the expression of multiple Wnts and Fzs as well as downstream targets of the canonical Wnt signaling pathway, Dvl-1 and Lef-1, in the adult rodent ovary. Furthermore, several Wnts and Fzs are expressed at specific stages of ovarian follicular development, ovulation, and luteinization, suggesting specific functions for these signaling molecules in the mature ovary. Among the Wnts found to be expressed are Wnt-4, Wnt-7a, and Wnt-2, which have been shown to impact early reproductive development (8, 50, 51). The absence of an observed ovarian phenotype for Wnt-7a and Wnt-2 mutant mice may be because of redundancy of Wnt functions. Because several Wnt transcripts were found to be expressed in the adult ovary by RT-PCR analyses in our study, Wnts may have both distinct and overlapping roles during follicle growth, ovulation, and luteinization. The finding that multiple Fz receptors are also expressed in the ovary suggests that Wnts have the potential to activate more than one signaling pathway, such as the Wnt/ß-catenin or Wnt/Ca2+ pathways.

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{alpha}-hydroxylase (54), FSH receptor (18, 19), MIS (15, 16), and inhibin-{alpha} (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.


   Footnotes
 
This work was supported in part by Grants NIH-HD-16229 and SCCPRR-HD-07495 (to J.S.R.) and by CA-64851 (to N.M.G.).

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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[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]