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Department of Molecular and Cellular Biology, Baylor College of Medicine (M.H., J.S.R.), Houston, Texas 77030; Research and Development, NV Organon (S.M.M.), 5340 BH Oss, The Netherlands; University of Western Australia School of Anatomy and Human Biology (A.D.), Crawley, Western Australia 6009, Australia; West Australian Institute of Medical Research, Sir Charles Gairdner Hospital (A.D.), Shenton Park, Western Australia 6009, Australia; and Department of Clinical Research, University of Bern (R.R.F.), CH-3010 Bern, Switzerland
Address all correspondence and requests for reprints to: Dr. 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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and ß) that are obligatory for normal follicle maturation (3), the progesterone receptor (PR) that is essential for ovulation (4), and members of the TGFß superfamily (e.g. Mullerian inhibitory substance, growth differentiation factor-9, and bone morphogenic protein-4, -7, and -15) that impact follicle organization and ovarian cell functions (5, 6, 7, 8, 9, 10). In addition, Wnt-4, a member of the Wnt/wingless family of extracellular signaling proteins, is critical during early ovarian development. Mice null for Wnt-4 exhibit sex-reversed ovaries that are depleted of oocytes at birth and contain supporting cells expressing genes characteristic of testis development (11). These mice die shortly after birth, precluding further analysis of Wnt-4 function in the mature rodent ovary. Genotypic XY humans with a duplicated part of chromosome 1p that includes WNT-4 are feminized (12). Overexpression of Wnt-4 in cultured mouse Leydig and Sertoli cells resulted in strong up-regulation of Dax1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita critical region on the X chromosome, gene 1) (12), a putative repressor of steroidogenic factor-1, which in the adult gonad regulates the expression of many genes, including aromatase (13), P450 side-chain cleavage (14), Mullerian inhibitory substance (15), and FSH receptor (16, 17). Therefore, Wnt-4 (and possibly other Wnt proteins) may perform important functions in the adult ovary that remain to be defined. Recently, we and others reported the expression of several members of the Wnt and Frizzled (Fz) families as well as of downstream components of the Wnt-Fz signaling pathway in the mature rodent ovary (18, 19). Wnt-4, Fz-4, and Fz-1 were among the transcripts detected and found to be regulated by gonadotropins and steroids. Fz-1 mRNA was specifically induced by the LH surge in granulosa cells of periovulatory follicles, whereas transcripts for both Wnt-4 and Fz-4 were elevated in terminally differentiated luteal cells. Wnt-4, but not Fz-4, was also increased in granulosa cells of small preantral follicles. In ovaries of PR-null mice, Fz-1, but not Wnt-4, expression was reduced around the time of ovulation. Together these results suggest important roles for Wnt-Fz signals during ovarian follicular development, ovulation, and luteinization.
Wnt proteins comprise a large family of locally acting, extracellular signaling molecules that perform key roles during such processes as cell fate specification, proliferation, differentiation, and tissue patterning (20, 21). Wnts activate distinct signaling pathways, including the canonical Wnt signaling pathway that is initiated by formation of a complex between Wnt, a seven-transmembrane receptor of the Fz family, and either LRP5 or LRP6 (22, 23). Activation of the canonical pathway leads to downstream signaling via stabilization of ß-catenin, whereas noncanonical Wnt pathways lead to c-Jun N-terminal kinase activation or increased intracellular Ca2+ levels, activation of protein kinase C and Ca2+/calmodulin-dependent protein kinase II, and decreased cGMP levels (24, 25, 26, 27, 28).
A family of secreted glycoproteins called secreted Fz-related proteins (sFRPs) also impacts Wnt-Fz signaling. The sFRPs are approximately 300 amino acids in length, contain an N-terminal cysteine-rich domain (CRD) homologous to the putative Wnt-binding site of Fzs, and a C-terminal domain containing a netrin-like module (29, 30). Several studies provide evidence for direct binding between Wnts and sFRPs as well as modulation of Wnt signals by sFRPs (31, 32, 33). The interaction between Wnts and sFRPs is thought to be mediated by the CRD, although a sFRP-1 mutant lacking the CRD retained the ability to bind Wg, the Drosophila ortholog of mammalian Wnt-1 (32). The CRD of Hfz6 and human FRP were also shown to interact (31), providing another mechanism by which sFRPs may regulate Wnt signals.
Of interest to our studies, sFRP-4 (also called frpAP, DDC-4, FrpHE, and FrzB-2) has been found to be expressed in the rat ovary (34). The studies described herein were undertaken to determine in greater detail the hormone-regulated (peptide and steroid) expression of sFRP-4, and its cell-specific expression in rodent ovaries in relation to other Wnt-Fz signals. The results of RT-PCR and in situ hybridization assays revealed increased expression of sFRP-4 mRNA in response to the LH surge in granulosa cells of periovulatory follicles and in terminally differentiated luteal cells. sFRP-4 expression does not appear to be PR regulated, but is induced by LH and maintained in corpora lutea by PRL. The localization of sFRP-4 to periovulatory follicles and corpora lutea overlaps with sites of Fz-1, Wnt-4, and Fz-4 expression, suggesting potential regulation of these signaling pathways by sFRP-4.
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Female Sprague Dawley rats (Harlan Sprague Dawley, Inc.) were hypophysectomized (H) on d 26 of age and given the following hormonal regimen beginning 4 d later. H rats were injected sc with 1.5 mg 17ß-estradiol/0.2 ml propylene glycol (Sigma-Aldrich Corp., St. Louis, MO) once daily for 3 d (HE). HE rats then received sc injections of 1.0 µg/0.1 ml ovine FSH (NIH oFSH-16, National Hormone and Pituitary Program, Rockville, MD) twice daily for 2 d (HEF), followed by a single ip injection of 10 IU hCG (HEF/hCG; Organon Special Chemicals). In both the mouse and rat models, ovulation occurs approximately 12–14 h after hCG administration. To examine corpora lutea function, luteinization was induced in additional HEF rats that were given a single sc injection of 10 IU hCG with or without sc injections of 100 µg ovine PRL (National Hormone and Pituitary Program) prepared as previously described (35) and given twice daily for 2–4 d.
PR knockout (PRKO) mice (provided by Dr. J. Lydon, Baylor College of Medicine, Houston, TX) fail to ovulate in response to hCG (4, 36). These mice were also injected with PMSG and hCG as described above to induce follicle growth and differentiation. 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
Whole ovaries (WO) were isolated from PMSG/hCG-stimulated wild-type or PRKO mice at selected time intervals as well as from d 15 pregnant C57BL/6 mice (Harlan Sprague Dawley, Inc.) and were used for extraction of total RNA using TRIzol reagent (Invitrogen/Life Technologies, Carlsbad, CA). The RNAs were then purified as specified by the manufacturer. Total RNA was similarly prepared from whole ovaries of HEF/hCG rats stimulated for 24, 48, 72, and 96 h, from WO of HEF/hCG+PRL rats stimulated for 48, 72, and 96 h and from WO of intact immature Sprague Dawley rats (23–25 d old; Harlan Sprague Dawley, Inc.). RNA was also extracted from granulosa cells and residual cells isolated from H, HE, HEF, and HEF/hCG 12 h rat ovaries. Briefly, granulosa cells were harvested from ovaries by needle puncture as previously described (37, 38) in DMEM/nutrient mixture F-12 (Invitrogen/Life Technologies) containing penicillin (100 U/ml) and streptomycin (100 µg/ml; Sigma-Aldrich Corp.), pelleted by centrifugation at low speed to remove the medium, and homogenized in TRIzol reagent to extract total RNA. Residual cells that consist of the remaining ovarian cell types, including thecal cells, interstitial cells, endothelial cells, and trace granulosa cells, that were not easily isolated from small preantral follicles by the puncture method were also homogenized in TRIzol reagent for extraction of RNAs.
Total RNA (300 ng) was reverse transcribed using poly-deoxythymidine (Amersham Pharmacia Biotech, Piscataway, NJ) 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 into two aliquots. Primers for mouse sFRP-4 (forward, 5'-CATCAAGCCCTGCAAGTCTG-3'; reverse, 5'-TAAGGGTGGCTCCATCACAG-3') or rat sFRP-4 (forward, 5'-TATGACCGTGGAGTGTGCAT-3'; reverse, 5'-CTTAGGACTGGCAGGTTTGG-3') were added to one aliquot, and primers for ribosomal protein L-19 (36, 39) were added to the other aliquot as an internal control. [32P]Deoxy-CTP (ICN, Los Angeles, CA), Taq polymerase, and Thermocycle buffer (Promega Corp.) were also included in the PCRs. Mouse sFRP-4, rat sFRP-4, and L-19 were amplified in 18, 23, and 20 PCR cycles (94 C for 1 min, 60 C for 2 min, 72 C for 3 min), respectively. The amplified cDNA products were resolved on a 5% polyacrylamide gel and subsequently quantified using a Storm 860 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). The identities of the PCR products were verified by sequencing (as described below).
In situ hybridization
The RT-PCR products for mouse and rat sFRP-4 were subcloned into the pCR4-TOPO vector (TOPO TA Cloning Kit, Invitrogen/Life Technologies), verified by sequencing, and used to produce [35S]UTP-labeled antisense and sense riboprobes using the Riboprobe In Vitro Transcription Systems Kit (Promega Corp.). In situ hybridization was performed as described by Wilkensen (40) and as previously shown in our laboratory (18, 41). Briefly, ovaries from PMSG/hCG-stimulated mice and hormone-treated H rats were isolated at selected time intervals, fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned at 7 µm onto Fisherbrand SuperFrost Plus microscope slides (Fisher Scientific, Pittsburgh, PA). Tissue sections were deparaffinized, rehydrated, treated with 20 µg/ml proteinase K and 0.1 M triethanolamine/acetic anhydride, and dehydrated before overnight incubation with radiolabeled probe at 55 C. Next, slides were washed under highly stringent conditions and dried. The specificity and intensity of the probe were determined by exposing slides overnight to X-OMAT film (Eastman Kodak, Rochester, NY). Afterward, each slide was dipped in photographic NTB-2 emulsion (Eastman Kodak) and exposed at 4 C. Slides were developed with D-19 developer and fixer (Eastman Kodak) and were stained with hematoxylin. Light- and dark-field illuminations were used to visualize tissue histology and the mRNA probe, respectively.
Immunohistochemistry
Rat ovaries were fixed, embedded, and sectioned as described above. Tissue sections were deparaffinized in xylenes, quenched with 3% H2O2 in methanol, and rehydrated. Slides were next incubated in 10 mM sodium citrate (pH 6.0) at 90 C for 20 min, removed from heat, cooled at room temperature for 20 min, and washed in PBS (80 mM Na2HPO4, 20 mM NaH2PO4, and 100 mM NaCl). Sections were blocked with 20% goat serum in PBS/0.025% Tween 20 for 1 h at room temperature, then incubated overnight at room temperature with affinity-purified polyclonal rabbit anti-sFRP-4 antibody (42) diluted 1:200 in 10% goat serum (in PBS/0.025% Tween 20). The next day, sections were washed in PBS/0.025% Tween 20 and incubated for 1 h at room temperature with biotinylated antirabbit IgG (Vector Laboratories, Inc., Burlingame, CA) diluted 1:450 in 10% goat serum (in PBS/0.025% Tween 20). Afterward, sections were washed first in PBS/0.025% Tween 20, then in PBS alone. After the washes, sections were incubated for 30 min at room temperature with streptavidin-conjugated peroxidase diluted 1:500 in PBS, then washed again in PBS. Localization of the primary antibody was visualized with diaminobenzidine (Vector Laboratories, Inc.), which produces a brown stain. Sections were dehydrated, cleared in xylenes, and mounted with Permount (Fisher Scientific).
Immunofluorescence
Immature (23–25 d old) female Sprague Dawley rats were injected sc with a low dose of hCG (0.15 IU) twice daily for 2 d to promote the development of preovulatory follicles, followed by a single ip injection of 10 IU hCG the next morning to initiate luteinization (35). After 7 h, ovaries were collected, and large vascular periovulatory follicles were dissected from the ovaries. Granulosa cells were harvested from the isolated follicles by the needle puncture method and cultured on glass coverslips for 4 d in DMEM/Ham’s F-12/1% fetal bovine serum [FBS; Invitrogen/Life Technologies and Hyclone (Logan, UT)] containing penicillin (100 U/ml) and streptomycin (100 µg/ml; Sigma-Aldrich Corp.). These cells spontaneously luteinize and constitutively express luteal cell markers such as P450scc (35). Additional luteinizing granulosa cells were cultured in medium with or without PRL (1 µg/ml) for another 3 d. Granulosa cells from nonovulatory follicles were also isolated from the remainder of the ovaries by needle puncture and similarly cultured.
After culture, granulosa and luteinized granulosa cells were fixed in 4% formaldehyde for 30 min at room temperature. After washing with PBS to remove the fixative, cells were permeabilized in 0.5% Nonidet P-40/PBS, washed with PBS, and blocked in 4% BSA/PBS for 1 h at room temperature. Next, cells were incubated overnight at 4 C with rabbit anti-sFRP-4 antibody (42) diluted 1:200 in 4% BSA/PBS. On the following morning, cells were washed in PBS and incubated in the dark for 1 h at room temperature with fluorescein goat antirabbit Ig (Vector Laboratories, Inc.; 1:20 in 4% BSA/PBS). Afterward, cells were washed in PBS and mounted onto slides with Vectashield mounting medium (Vector Laboratories, Inc.). The edges of the coverslips were sealed with nail polish.
Western blot
Granulosa cells from ovulatory and small follicles were isolated and cultured in six-well dishes as described for immunofluorescence. Protein was extracted from nonluteinized and luteinized granulosa cells in boiling sodium dodecyl sulfate (SDS) buffer [100 mM Tris (pH 6.8), 2% SDS, 20% glycerol, 10% 2-mercaptoethanol, and a pinch of bromphenol blue). Medium was also collected from these same cultures and concentrated using Centricon-30 microconcentrators (Amicon, Beverly, MA). SDS sample buffer [0.35 M Tris-Cl/0.01 M SDS (pH 6.8), 30% glycerol, 10% SDS, 6% 2-mercaptoethanol, and 0.012% bromphenol blue] was added to an aliquot of concentrated medium and boiled before loading samples onto an acrylamide gel. Protein samples 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) for 1 h at room temperature, then incubated with affinity-purified polyclonal rabbit anti-sFRP-4 antibody (1:1,000 in 5% milk) (42) also for 1 h at room temperature. Next, blots were washed in Tris-buffered saline containing 0.5% Tween 20 and incubated for 1 h at room temperature with antirabbit IgG peroxidase-linked antibody (Amersham Pharmacia Biotech) diluted 1:10,000 in 5% milk. After washing blots in Tris-buffered saline containing 0.5% Tween 20, detection of immunoreactive protein was performed using SuperSignal chemiluminescent detection reagents (Pierce Chemical Co., Rockford, IL), and protein was visualized by autoradiography.
Granulosa cell cultures
Granulosa cells were isolated by needle puncture (37, 38) from intact immature rats (23–25 d of age) treated with estradiol (E; 1.5 mg 17ß-estradiol/0.2 ml propylene glycol; Sigma-Aldrich Corp.) once daily for 3 d to promote follicle growth. Granulosa cells were also isolated from immature rats treated with 10 IU PMSG (Professional Compounding Center of America) for 48 h to bring follicles to the preovulatory phenotype (43). E- and PMSG-primed granulosa cells were seeded at a density of 1 x 106 cells/well in a six-well plate. E-primed granulosa cells, which were seeded in serum-coated wells, were cultured overnight in serum-free medium (DMEM/F-12/penicillin/streptomycin), followed by the addition of FSH (50 ng/ml) and testosterone (T; 10 ng/ml) to promote granulosa cell differentiation, and forskolin (Fo; 10 µM) and/or phorbol 12-myristate 12-acetate (PMA; 20 µM) to stimulate acute agonist effects. PMSG-primed granulosa cells were cultured overnight in medium containing 5% FBS. On the next day, the medium was removed, and cells were washed with PBS and refed with serum-free medium containing the above hormones or agonists. Granulosa cells were harvested in TRIzol reagent (Invitrogen/Life Technologies) at selected times after the addition of hormones or agonists for preparation of RNA as described above.
Statistics
ANOVA was performed to determine whether there was a significant difference between sample values within an experiment. To more clearly determine where those differences occurred, the Tukey test was performed, and t test confirmed the significance of the differences. P < 0.05 was considered significant. All P values are provided in the figure legends.
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). sFRP-4 mRNA levels increased significantly in response to stimulation with hCG between 2 and 4 h. After a decrease at 24 h, sFRP-4 message increased again at 48 h after hCG to levels observed between 4 and 16 h. The increased presence of sFRP-4 in ovaries containing corpora lutea 48 h after hCG is supported by elevated levels in d 15 pregnant mouse ovaries that contain highly functional corpora lutea.
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). Between 4 and 12 h after hCG stimulation, sFRP-4 expression was markedly elevated in granulosa cells of large follicles destined for ovulation. sFRP-4 mRNA levels were also elevated in corpora lutea formed 48 h after hCG and in corpora lutea of d 15 pregnant mouse ovaries.
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), suggesting that sFRP-4 is not a PR-regulated gene in the ovary.
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). Expression increased after stimulation with FSH for 48 h and was highly induced in granulosa cells after 12 h hCG (HEF/hCG12 h). sFRP-4 mRNA levels were low in residual cells of H and HE rat ovaries. A small increase in expression was observed in residual tissue of HEF and HEF/hCG12 h rat ovaries, probably due to the presence of granulosa cells that were not easily isolated from small follicles by the puncture procedure. In contrast to the high expression observed in mouse ovaries stimulated for 48 h with hCG, sFRP-4 expression appeared to decline in HEF rat ovaries isolated after 24 and 48 h of hCG stimulation. This may be due to the absence of PRL signals that are normally received from the pituitary and are important for the maintenance of functional corpora lutea in rodents.
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, sFRP-4 expression levels appeared low after 48 h of hCG (Fig. 4B). However, sFRP-4 message increased in ovaries exposed to PRL (HEF/hCG+PRL 48 h). Similarly, sFRP-4 transcript levels were significantly induced in ovaries that had been stimulated with hCG+PRL for 72 and 96 h compared with those in ovaries stimulated with hCG alone. These results strongly suggest that PRL impacts sFRP-4 expression in corpora lutea of H rats.
Localization of sFRP-4 transcripts to corpora lutea of PRL-stimulated and pregnant rat ovaries
In situ hybridization analyses were performed to localize sFRP-4 mRNA in HEF/hCG with or without PRL rat ovaries and in pregnant rat ovaries. sFRP-4 mRNA was not easily detected above background levels in ovaries of rats stimulated with HEF/hCG for 48 and 72 h (data not shown) and for 96 h (Fig. 5). However, in ovaries of HEF/hCG-treated rats that had also been stimulated with PRL for 48 and 72 h (data not shown) and 96 h, the sFRP-4 message in corpora lutea was elevated, confirming the above RT-PCR data. As expected, sFRP-4 mRNA was also evident in corpora lutea of ovaries from normal rats after 14 d of pregnancy (Fig. 5) as well as after 7 and 20 d of pregnancy (data not shown).
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). After 12 h of FSH/T, a 2.5-fold increase in sFRP-4 expression was observed over the control value, and sFRP-4 mRNA was further induced approximately 40- and 70-fold over control levels after 24 and 48 h of FSH/T, respectively, when cells had reached a more differentiated phenotype (Fig. 6A). The addition of Fo/PMA for 2 h after 48 h of FSH/T did not significantly increase sFRP-4 expression levels above those observed in granulosa cells after 48 h of FSH/T. Control unstimulated granulosa cells after 48 h in culture did not express detectable levels of sFRP-4 message.
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). A 2- to 3-fold increase in sFRP-4 expression was observed after 4- and 8-h culture of cells in the presence of Fo alone or FSH/T, and further induction (7- to 8-fold) occurred after 12 h with Fo alone or FSH/T (Fig. 6B). After differentiation of cells in the presence of FSH/T for 48 h, sFRP-4 was induced approximately 60-fold over control unstimulated granulosa cell levels. Addition of Fo alone or Fo/PMA for another 2 h after 48 h of FSH/T did not further induce sFRP-4. That PMA had no effect on sFRP-4 expression in differentiated cells indicates that the acute effect of protein kinase C alone does not further impact differentiation. Taken together, these results indicate that the expression of sFRP-4 by granulosa cells is dependent on hormone-induced differentiation of granulosa cells to a luteal-like phenotype.
Expression and localization of sFRP-4 protein
To localize sFRP-4 protein in rat ovaries, immunohistochemistry was performed using an affinity-purified polyclonal rabbit anti-sFRP-4 antibody (42). Staining for sFRP-4 protein was evident in granulosa cells of antral follicles and in luteal cells of pregnant rat ovaries, whereas little staining above background was detected in granulosa cells of small follicles (Fig. 7A). Furthermore, the intense staining for sFRP-4 within the cytoplasm of luteal cells indicated that this secreted protein was localized to secretory vesicles.
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, left). However, the immunosignal for sFRP-4 was clearly visible in luteinized granulosa cells as punctate regions throughout the cytoplasm, but not in the nucleus (Fig. 7B, middle and right). This pattern of staining within the cytoplasm is likely to be secretory vesicles containing the sFRP-4 protein. Western blot analysis was performed to confirm the presence of sFRP-4 protein in these cultured cells. sFRP-4 protein was detected in nonluteinized granulosa cells, but was much higher in luteinized granulosa cells (Fig. 7C). Interestingly, Western blot analysis of medium collected from these same cultures revealed the presence of sFRP-4 protein in the medium from luteinized granulosa cells, but not from nonluteinized granulosa cells that had also been stimulated with PRL (Fig. 7D). Thus, PRL appears to stimulate the secretion of sFRP-4 protein from luteinized granulosa cells. |
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A role for sFRP-4 in luteal cells is supported by the finding that its expression in these cells is regulated by the luteotropic hormone PRL. Stimulation of HEF/hCG rats additionally with PRL was effective in inducing and maintaining the expression of sFRP-4 transcripts in corpora lutea. Interestingly, the expression of sFRP-4 protein in luteinized granulosa cells cultured in the absence or presence of PRL is similar to the pattern observed previously for the expression of cholesterol side-chain cleavage cytochrome P450 (CYP11A). Once expression of CYP11A mRNA and protein as well as progesterone biosynthesis is induced by LH, CYP11A is constitutively expressed in luteinized granulosa cells in culture (35). PRL also regulated the secretion of sFRP-4 protein, as immunoreactive sFRP-4 was detected only in medium collected from luteinized granulosa cells cultured in the presence of PRL, but not in medium from cells cultured in the absence of PRL. Thus, PRL appears to stimulate not only the expression of sFRP-4 in corpora lutea in vivo, but also secretion of the protein from luteinized granulosa cells in culture. A potential role for Wnt/Fz regulation of steroidogenesis is indicated because Wnt-4 and Fz-4 are expressed in luteal cells, and gonads of female mice null for Wnt-4 (at birth) misexpress the steroidogenic enzymes 3ß-hydroxysteroid dehydrogenase and 17-hydroxylase that participate in progesterone and androgen biosynthesis, respectively (11). It is tempting to speculate that sFRP-4 may modulate Wnt-4 signals, possibly through the Fz-4 receptor, to impact steroidogenesis in corpora lutea. In addition or alternatively, sFRP-4/Wnt-4/Fz-4 may regulate or be targets of specific kinases that are highly expressed in luteal cells (Sgk, components of the p38 MAPK pathway) (45, 46, 47) or of other signaling pathways, such as the prostaglandin F2 pathway that is involved in parturition (48).
Regulation of sFRP-4 expression by LH and PRL may be direct, via specific regulatory elements contained within the promoter region (from -202 to -144) found to be essential for efficient transcription of sFRP-4 (49). Using TFSEARCH version 1.3 software, Yam et al. (49) identified binding sites for Stat3, Lyf-1, and myeloid zinc finger protein 1 in the region -196 to -185, and CCAAT/enhancer-binding protein-ß, GATA-1, and cAMP response element-binding protein in the region -149 to -141. In the ovary, CCAAT/enhancer-binding protein-ß mRNA is induced, whereas cAMP response element-binding protein is rapidly phosphorylated and activated, in granulosa cells in response to an ovulatory dose of LH/hCG (50, 51, 52). These LH-responsive factors may confer the rapid LH-dependent induction of sFRP-4 expression. During luteinization of granulosa cells, induction of sFRP-4 mRNA becomes responsive to PRL. PRL activates the Janus kinase signal transducer and activator of transcription (Stat) pathway that leads to phosphorylation of Stat proteins (Stat1, Stat3, and mainly Stat5) (53). Although both Stat3 and Stat5 are expressed in the ovary, Stat5b appears to be the principal target of PRL signals during luteinization (54). Stat3 is activated primarily in granulosa cells of small follicles by a cytokine pathway other than PRL (54). Thus, activated Stat5b may be binding directly to the putative Stat binding site in the sFRP-4 promoter and regulating sFRP-4 gene expression in the corpus luteum. The mechanism by which PRL regulates the secretion of sFRP-4 from luteal cells is not yet known.
Whether sFRP-4 functions as an antagonist or agonist of Wnt signals or performs some other novel function in the ovary is unclear. Although sFRPs have generally been described as antagonists of Wnt signals, recent studies provide evidence for positive regulation as well. During kidney development, Wnt-4 induces epithelial conversion of the metanephric mesenchyme (55). sFRP-1 blocks events associated with epithelial conversion (tubulogenesis and expression of lim-1, sFRP-2, and E-cadherin) in cultures of metanephroi, whereas concurrent treatment with sFRP-2 rescues some branching and tubular morphogenesis (33). Because sFRP-2 and Wnt-4 are coexpressed in newly formed epithelia, sFRP-2 may compete locally with sFRP-1 to promote Wnt-4 signaling (33). Recently, sFRP-1 has been observed in the mouse ovary (56). sFRP-4 and sFRP-1, then, may similarly compete or act together to modulate Wnt signals in the ovary. In another study, Üren et al. (32) reported biphasic regulation of wingless activity by sFRP-1, where low concentrations of sFRP-1 increased and high concentrations reduced armadillo (the Drosophila ortholog of ß-catenin) levels. Formation of sFRP homodimers or sFRP-Fz heterodimers may be important for this biphasic regulation. In vitro cotransfection of human FRP and the CRD domain of Hfz6 led to complex formation (31), and crystallographic data revealed the presence of a dimer interface in the crystals of both sFRP-3 and mFz8 CRDs (57), suggesting potential biological significance of CRD dimerization.
In addition to the N-terminal CRD domain, sFRPs have a C-terminal netrin-like (NTR) domain that is present in other proteins, including some complement proteins, type I procollagen C-proteinase enhancer proteins, and tissue inhibitors of metalloproteinases (TIMPs) (58). The NTR modules of TIMPs and possibly also of procollagen C-proteinase enhancer proteins are involved in the inhibition of extracellular matrix metalloproteinases (58). Interestingly, TIMP-1 expression, like that of Fz-1, is up-regulated in periovulatory follicles by the LH surge, and the expression of TIMPs varies with luteal formation, maintenance, and regression (18, 59). However, the role of the NTR domains of sFRPs is not yet known. Based on homology to metalloproteinase inhibitors, it is tempting to speculate that sFRPs may also be involved in extracellular matrix remodeling. Thus, in addition to modulating Wnt signals during follicle growth, ovulation, and luteinization, sFRP-4 may be important for regulating matrix formation or tissue breakdown during ovulation, and subsequent remodeling and differentiation to the corpus luteum.
In summary, the results presented herein document that sFRP-4 mRNA expression and protein secretion are regulated by hormones LH and PRL in the rodent ovary. Its elevated expression in luteinized granulosa cells in vivo and in vitro suggests that it impacts a Wnt/Fz pathway in these cells, possibly Wnt-4/Fz-4, which is coordinately expressed in luteal cells. Whether sFRP-4 enhances or antagonizes Wnt/Fz signaling in this ovarian tissue is not yet known.
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Abbreviations: CRD, Cysteine-rich domain; E, estradiol; FBS, fetal bovine serum; Fo, forskolin; Fz, Frizzled; H, hypophysectomized; hCG, human chorionic gonadotropin; HE, hypophysectomized and treated with 17ß-estradiol; HEF, hypophysectomized and treated with 17ß-estradiol and FSH; NTR, C-terminal netrin-like; PMA, phorbol 12-myristate 12-acetate; PMSG, pregnant mare serum gonadotropin; PR, progesterone receptor; PRKO, progesterone receptor knockout; SDS, sodium dodecyl sulfate; sFRP-4, secreted frizzled-related protein-4; Stat, signal transducer and activator of transcription; T, testosterone; TIMP, tissue inhibitors of metalloproteinase; WO, whole ovaries.
Received January 9, 2003.
Accepted for publication June 27, 2003.
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 C. M. Wayne, H.-Y. Fan, X. Cheng, and J. S. Richards Follicle-Stimulating Hormone Induces Multiple Signaling Cascades: Evidence that Activation of Rous Sarcoma Oncogene, RAS, and the Epidermal Growth Factor Receptor Are Critical for Granulosa Cell Differentiation Mol. Endocrinol., August 1, 2007; 21(8): 1940 - 1957. [Abstract] [Full Text] [PDF]
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J. R. A. Sherwin, A. M. Sharkey, P. Cameo, P. M. Mavrogianis, R. D. Catalano, S. Edassery, and A. T. Fazleabas Identification of Novel Genes Regulated by Chorionic Gonadotropin in Baboon Endometrium during the Window of Implantation Endocrinology, February 1, 2007; 148(2): 618 - 626. [Abstract] [Full Text] [PDF]
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D. P. Hewitt, P. J. Mark, A. M. Dharmarajan, and B. J. Waddell Placental Expression of Secreted Frizzled Related Protein-4 in the Rat and the Impact of Glucocorticoid-Induced Fetal and Placental Growth Restriction Biol Reprod, July 1, 2006; 75(1): 75 - 81. [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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S. A. Pangas, X. Li, E. J. Robertson, and M. M. Matzuk Premature Luteinization and Cumulus Cell Defects in Ovarian-Specific Smad4 Knockout Mice Mol. Endocrinol., June 1, 2006; 20(6): 1406 - 1422. [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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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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R. S. McRae, H. M. Johnston, M. Mihm, and P. J. O'Shaughnessy Changes in Mouse Granulosa Cell Gene Expression during Early Luteinization Endocrinology, January 1, 2005; 146(1): 309 - 317. [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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M. Jo and T. E. Curry Jr Regulation of Matrix Metalloproteinase-19 Messenger RNA Expression in the Rat Ovary Biol Reprod, December 1, 2004; 71(6): 1796 - 1806. [Abstract] [Full Text] [PDF]
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) and PRKO mouse ovaries (
). No significant differences were observed between the PR heterozygote and PRKO mouse ovarian samples. RNA was prepared separately from ovaries of three individual PR heterozygote or PRKO mice after PMSG or PMSG/hCG treatment at each time interval, and the results were analyzed separately by RT-PCR. Data are presented as the mean ± SEM of phosphorimage-quantitated sFRP-4 expression normalized to L19.
) compared with levels in ovaries of HEF/hCG rats (
40- to 70-fold over control levels) in granulosa cells that had differentiated in the presence of FSH/T for 24 and 48 h (P < 0.05 and P < 0.01, respectively). B, sFRP-4 message was also examined in granulosa cells isolated from ovaries of PMSG-stimulated rats that contain large preovulatory follicles. sFRP-4 mRNA was low in control and agonist-treated (Fo or Fo/PMA) or hormone-treated (FSH/T) cells at 2 h. sFRP-4 expression increased progressively over time with agonist or hormone treatment, and a 7- to 8-fold induction was observed after 12 h Fo or FSH/T (P < 0.05). Expression was most highly induced (