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Endocrinology Vol. 146, No. 9 4127-4137
Copyright © 2005 by The Endocrine Society

Multiple Signaling Defects in the Absence of RIP140 Impair Both Cumulus Expansion and Follicle Rupture

Jennifer M. A. Tullet, Victoria Pocock, Jennifer H. Steel, Roger White, Stuart Milligan and Malcolm G. Parker

Institute of Reproductive and Developmental Biology, Imperial College London (J.M.A.T., J.H.S., R.W., M.G.P.), London W12 0NN, United Kingdom; and Kings College London (V.P., S.M.), London SE1 1UL, United Kingdom

Address all correspondence and requests for reprints to: Dr. Malcolm G. Parker, Institute of Reproductive and Developmental Biology, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, United Kingdom. E-mail: m.parker{at}imperial.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The nuclear receptor corepressor RIP140 is essential in the ovary for ovulation, but is not required for follicle growth and luteinization. To identify genes that may be subject to regulation by RIP140 or play a role in ovulation, we compared ovarian gene expression profiles in untreated immature wild-type and RIP140 null mice and after treatment with pregnant mare serum gonadotropin and human chorionic gonadotropin. Many genes involved in signaling, extracellular matrix formation, cell-cell attachment, and adhesion were aberrantly regulated in the absence of RIP140, varying according to the hormone status of the mice. Notable among these was the reduced expression of a number of genes that encode components of signaling pathways and matrix proteins required for cumulus expansion, a key remodeling process necessary for ovulation. Histological analysis confirmed that cumulus expansion in RIP140 null mice is reduced, oocyte detachment from the mural cell wall is impaired, and follicles fail to rupture in response to LH. Although the expression of many genes involved in cumulus cell expansion was reduced, there was a subset of genes involved in extracellular matrix formation and cell-cell interactions that was up-regulated and may interfere with ovarian tissue remodeling. We propose that widespread gene dysregulation in ovarian tissues in the absence of RIP140 leads to the anovulatory phenotype. This helps to define an important role for RIP140 in the regulation of multiple processes leading to ovulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OVULATION IS A critical process in mammalian reproduction. The LH surge initiates a dramatic, coordinated cascade of hormonal and biochemical changes that eventually lead to the release of the oocyte and luteinization of the remaining follicle. The morphological events before ovulation include the resumption of meiosis in oocytes, expansion of the cumulus-oocyte complex, reprogramming of the granulosa cells to luteinize, vascular responses in the theca and interstitial tissues, and, finally, follicular rupture with release of the oocyte and its surrounding cumulus cells (1, 2). Many of these changes are consistent with the idea of ovulation resembling an inflammatory process and are accompanied by the recruitment and invasion of leukocytes and macrophages (3, 4).

Although all of the above events are initiated by the preovulatory LH surge, multiple signaling cascades are involved (5, 6). The availability of genetically modified mice with specific gene defects has provided a tremendous impetus to unravel the regulatory pathways (6, 7, 8). Many of the genes identified as having key roles in the ovulatory pathways encode proteins with relatively well-defined signaling functions, including the progesterone receptor (9); the epidermal growth factor ligands, amphiregulin, betacellulin, and epiregulin (10, 11); and cyclooxygenase-2 (COX-2) (12, 13). Ovulation fails to occur in mice lacking either the progesterone receptor or COX-2, reflecting the critical role for these proteins in mediating the events following the LH surge (9, 12). Partial failure to ovulate occurs in mice lacking genes coding for some of the structural proteins and signals involved in cumulus cell expansion; these include TNF-stimulated gene 6 (TSG-6), bikunin [the light chain of inter-{alpha}-inhibitor (I{alpha}I)], pentraxin-3, hyaluronic acid 2, versican, the prostaglandin EP2 receptor, and phosphodiesterase D4 (10, 14, 15, 16, 17). The partial ovulatory response observed in such animals may reflect a degree of redundancy or compensation in the signaling pathways involved.

We have identified another gene, RIP140 (18), which is critical for ovulation (19). RIP140 acts as a corepressor of the ligand-dependent family of nuclear receptors (18, 20, 21). Our initial observations indicated that RIP140 null females are infertile due primarily to a failure to ovulate, with their ovaries showing retained oocytes in otherwise normal corpora lutea (19). Ovarian transplant experiments demonstrated that this phenotype results from a defect in the ovary, rather than as a result of defective hypothalamic-pituitary signaling (22).

In the present study we analyze the structural alterations and molecular mechanisms underlying the ovulatory defect caused by the absence of RIP140. We use expression profiling analysis to demonstrate that the anovulatory phenotype in RIP140 null mice is associated with the disruption of a number of the very early signaling cascades linking the preovulatory LH surge with cumulus expansion and, in addition, identify a number of other genes that may contribute to defective ovulatory responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and superovulation
The generation of RIP140 null mice has been previously described (19). Mice used in this study were backcrossed six generations to the C57BL/6J background, maintained under standard conditions with controlled light and temperature, and fed a chow diet. All experiments were performed according to Home Office guidelines. Immature (3–4 wk old) mice were injected ip either with 5 IU Folligon [pregnant mare serum gonadotropin (PMSG); Intervet, Milton Keynes, UK] alone or with PMSG followed 48 h later by an injection of 10 IU Chorulon [human chorionic gonadotropin (hCG); Intervet]. After the appropriate time period, ovaries were dissected out and immediately frozen in liquid nitrogen or were fixed and embedded in wax for serial sections. Tissue sections were prepared as described previously (23) and stained with hematoxylin and eosin.

Ovarian histology and cumulus measurements
Ovaries were fixed and embedded in paraffin wax. Serial sections (8 µm) were stained with hematoxylin and eosin, and five follicles were selected at random from each animal (four per treatment group) at each time point (0, 3, 6, and 9 h after hCG treatment). Individual follicles were tracked through the serial sections, and the maximum diameter of each follicle was measured. The cumulus-oocyte complexes within each follicle were examined and scored for signs of expansion and whether they were still obviously attached to the mural granulosa layers. The diameter of the cumulus was measured in the section containing the largest cross-section of the oocyte, with the edge being defined by the outermost cumulus cells on either side of the oocyte.

RNA extraction and expression analysis
Total RNA was isolated from the ovaries of individual animals using TRIzol reagent (Invitrogen Life Technologies, Inc., Carlsbad, CA), and cDNA was prepared as previously described (23). The expression of RIP140 and L-19 was determined with specific primers and TaqMan probes, and the expression of all other genes was determined using the SYBR Green reagent and gene-specific primers. Expression levels for all genes were normalized against the expression of the ribosomal coding gene L-19. Primer sequences can be obtained on request.

Affymetrix microarray hybridization and data analysis
Equal quantities of RNA were pooled from animals that had been judged to have responded appropriately to the hormonal treatments by gene expression analysis. Each group contained ovarian RNA from three animals, except for the RIP140 null ovaries treated with either PMSG or PMSG plus hCG, which (because of limited availability) each consisted of RNA from two animals. First-strand cDNA synthesis was performed using a total of 10 µg RNA with a T7-(deoxythymidine)24 primer and SuperScript II (Invitrogen Life Technologies, Inc.; 42 C, 1 h). Second-strand synthesis was carried out using Escherichia coli DNA ligase, Pol I, and ribonuclease H (Invitrogen Life Technologies, Inc.; 16 C, 2 h). After clean up (GeneChip Sample Cleanup Module; Affymetrix, Santa Clara, CA), the Bioarray High Yield RNA transcript Labeling Kit (Enzo Biochem, Farmingdale, NY) was used according to the manufacturer’s instructions to synthesize biotin-labeled antisense cRNA. Fragmentation and hybridization of the cRNA to the Affymetrix GeneChip Mouse Genome 430_2.0 arrays were carried out according to the manufacturer’s instructions. Additional details are available at the Medical Research Council/Clinical Sciences Centre/Imperial College Microarray Centre web site (http://microarray.csc.mrc.ac.uk). Data analysis was performed using the DNA-Chip analyzer (dChip, version 1.3) software package (24). The arrays were normalized, and the PM/MM difference model was used to calculate expression values. Hierarchical clustering analysis was performed to arrange genes according to similarity in pattern of expression (25). All microarray data obtained in this study from wild-type, RIP140 heterozygote, and RIP140 null ovaries is available from EMBL-EBI (Hinxton, UK) at www.ebi.ac.uk/arraysexpress.

Analysis of gene expression in distinct cell populations using laser capture microdissection
Ovaries were embedded in Tissue-Tek (Miles, Elkhart, IN) and frozen in liquid nitrogen. Cryosections (10 µm thick) were cut and mounted onto ribonuclease-treated membrane slides (P.A.L.M. Microlaser Technologies AG, Bernried, Germany). Sections were fixed briefly (10 sec) in 70% ethanol and counterstained with hematoxylin. Each population of ovarian cells (mural granulosa cells, cumulus granulosa cells with oocyte, and residual ovary containing thecal cells) was isolated from these sections using the P.A.L.M. Robot-Microbeam version 4.0 (P.A.L.M. Microlaser Technologies AG). Laser capture was performed with an approximately 15- to 30-µm laser beam, a laser power of 50 mV, and a laser power duration of 4–6 msec. Cells were collected in 2 µl mineral oil in the cap of a 0.5-ml tube and were stored on ice until RNA extraction. RNA was extracted within 2 h of cell collection using the P.A.L.M RNA extraction kit according to the manufacturer’s instructions. All RNA extracted was used for the RT reaction (Invitrogen Life Technologies, Inc.) as described above. Gene expression analysis was carried out as described above, and gene expression was normalized to L-19.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression profiling of ovarian gene expression in RIP140 null mice
RIP140 null mice fail to ovulate in response to ovulatory hormones. To identify genes aberrantly expressed in the ovaries of RIP140 null mice, expression profiling analysis was performed using Affymetrix oligonucleotide microarrays. RNA was isolated from the ovaries of mice that had been treated with hormones to induce follicle growth and ovulation. This comprised treatment with PMSG for 48 h or with PMSG plus hCG for 3 h, respectively. The RNA samples were initially examined for expression of the LH receptor and the progesterone receptor by quantitative PCR to act as markers for a successful response to the superovulatory hormone regimen. As expected, the mRNA for the LH receptor was increased more than 20-fold after PMSG treatment, whereas that for the progesterone receptor was similarly stimulated after hCG treatment (data not shown). Equal quantities of ovarian RNA from at least two mice (see Materials and Methods) were pooled, and the corresponding biotinylated RNA probes were synthesized. The expression analysis was performed using mouse full genome 430_2.0 oligonucleotide arrays. These arrays are comprised of 45,000 probe sets that represent 39,000 RNA transcripts, including those of 34,000 well-characterized genes.

The expression of each probe set on the array was then compared in wild-type and RIP140 null ovaries from immature, PMSG-treated, and PMSG- plus hCG-treated mice using dChip software. The total number of probe sets that were differentially expressed in ovaries from null mice was surprisingly large, with more than 1000 altered by at least 3-fold in each treatment (Fig. 1AGo). Consistent with the function of RIP140 as a transcriptional repressor, more probe sets were up-regulated than down-regulated in the RIP140 null mice (60% in untreated ovaries, 62% after PMSG treatment, and 64% after hCG treatment). A Venn diagram (Fig. 1BGo) summarizes the number of probe sets that were differentially expressed in more than one treatment group. The total number of differentially expressed probe sets was greatest after hCG treatment, when the defect in ovarian function becomes manifest. Approximately 200 of the probe sets up-regulated in response to PMSG and 250 probe sets of those increased after hCG treatment have reasonably well-described functions. In addition to those implicated in signaling cascades, transcriptional regulation, and enzyme functions, an appreciable proportion is implicated in cell-cell interactions and matrix attachment or formation (Fig. 1CGo).



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  		FIG. 1. Removal of RIP140 results in a trend toward up-regulated gene expression. The expression of probe sets from wild-type (WT) and RIP140 null [knockout (KO)] ovaries (untreated, after PMSG treatment, or after PMSG plus hCG treatment) were directly compared using dChip software. The numbers of probe sets (both known and unknown) dysregulated more than 3-fold in the RIP140 null ovaries after each hormone treatment were recorded. B, The combined comparison function of the dChip software was then used to determine the numbers of these dysregulated probe sets that were common to all hormone treatments. C, Genes up-regulated more than 3-fold with a defined function in the extracellular matrix or cell-cell adhesion were then subjected to hierarchical clustering analysis.

 
Although any of the many observed changes in gene expression in RIP140 null mice may contribute to the anovulatory phenotype, those involving the extracellular matrix are of particular interest due to the dramatic tissue remodeling that occurs before and during ovulation. Figure 1CGo indicates that many of these extracellular matrix genes are expressed at very low levels in wild-type animals, and that expression is induced in RIP140 null ovaries in response to hormone stimulation. In addition, these studies identify another group of genes that is down regulated in wild-type ovaries in response to hCG, but up-regulated in RIP140 null animals. To support and extend the array data, we analyzed the expression of six specific genes in this category [desmocollin-2, syndecan-4, cartilage acidic protein, stabilin-2, coronin actin-binding protein 1C, and serine protease inhibitor (serpin) A3N] using quantitative PCR of individual ovarian RNA samples from at least three wild-type and RIP140 null mice (Fig. 2Go). This confirmed that the mRNAs for the selected genes were up-regulated in a hormone-dependent manner in mice devoid of RIP140.



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  		FIG. 2. Genes involved in the extracellular matrix and cell-cell adhesion are up-regulated in RIP140 null mice. A, RNA was isolated from the ovaries of immature wild-type (WT) and RIP140 null [knockout (KO)] mice after no treatment, 48 h after PMSG injection, or 3 h after hCG treatment (given 48 h after PMSG). The expressions of six genes identified in Fig. 1CGo were assessed using quantitative PCR. The expression level of each gene is normalized against the expression of L-19, and each bar represents the combined average expression of that gene for at least three animals, with the error bars representing the SEM for that group. Each group included all samples included in the array pool plus additional samples that had also responded to the hormone treatments.

 
We then focused on genes with a role in ovarian function using the Ovarian Kaleidoscope Database (26), which documents the biological function, expression pattern, and regulation of genes in the ovary. This indicated that several such genes were down-regulated in RIP140 null animals in response to gonadotropin stimulation: 17 genes of a total 287 known genes were down-regulated more than 3-fold in RIP140 null mice after PMSG alone, and 30 genes were down-regulated after hCG treatment (Fig. 3Go). The list of dysregulated genes reflects the range of signaling, transcription, and enzymatic remodeling processes occurring in the ovary during follicular growth and the preovulatory period. It was noteworthy that this group included several genes with previously reported roles in the process of cumulus expansion (Fig. 3Go).



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  		FIG. 3. RIP140 null [knockout (KO)] mice fail to express genes vital for cumulus cell expansion. WT, Wild type. An ovarian database was mined for all of the down-regulated, known genes identified in Fig. 1AGo after PMSG or PMSG plus hCG treatment. Genes with recorded expression or function in the ovary were then subjected to hierarchical clustering analysis. Genes with a defined role in the process of cumulus cell expansion are highlighted in red.

 
RIP140 null ovaries have defects in cumulus expansion and follicle rupture
The anovulatory phenotype of RIP140 null mice was investigated by histologically examining the follicular structure in immature mice during the course of a hormonal stimulation regimen (PMSG, followed 48 h later by hCG). Serially sectioned ovaries were examined for Graafian follicle size, cumulus cell expansion, germinal vesicle breakdown, and follicular rupture.

Forty-eight hours after PMSG injection, follicles of wild-type and RIP140 null mice were similar in both size (diameters of 390 ± 8.8 and 377 ± 8.8 mm, respectively) and histology. Follicle diameters were also similar 9 h after hCG treatment (479 ± 17.1 and 499 ± 12.9 mm, respectively). Germinal vesicle breakdown appeared normal in both wild-type mice and mice devoid of RIP140 and had occurred within 3 h of hCG treatment. By 6 h after hCG treatment, the process of cumulus expansion had been initiated in all genotypes, but the degree of expansion was different between the wild-type and RIP140 null ovaries. Cumulus expansion was evident in over 90% of the follicles in wild-type animals, but in only 70% of the follicles in RIP140 null animals (Fig. 4AGo and data not shown). In addition, in RIP140 null follicles that demonstrated cumulus expansion, the degree of expansion was significantly smaller than that in the wild-type mice; the diameter of the oocyte-cumulus mass was 138 ± 3.9 vs. 154 ± 5 mm, respectively, 6 h after hCG treatment (P < 0.05) and 148 ± 6.7 vs. 190 ± 9 mm 9 h after hCG treatment (P > 0.001; data not shown). We then examined the ovaries for evidence of hyaluronic acid (the major component of the extracellular matrix associated with expansion of the cumulus-oocyte complex) 10 h after hCG treatment. Wild-type ovaries exhibited strong staining for hyaluronic acid in and around cumulus cells and at the antral edge of mural granulosa cells. This staining was eliminated by predigestion with hyaluronidase, indicating that the stain was specific. Staining specific for hyaluronic acid was also detectable around cumulus cells that had undergone partial expansion in RIP140 null ovaries, but was absent in follicles in which cumulus expansion had not occurred (data not shown).



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  		FIG. 4. Mice devoid of RIP140 demonstrate defective cumulus cell expansion. A, Representative sections from ovaries from wild-type (i; WT) and RIP140 null (ii; treated with PMSG for 48 h plus hCG for 10 h). Ovaries were paraffin fixed, sectioned, and stained with hematoxylin and eosin. B, Scatter graph showing individual diameters of cumulus oocyte complexes, and whether they were detached from the mural granulosa layer in wild-type, heterozygote (HET), and RIP140 knockout (KO) mice treated as described in A (n = 5 cumulus oocyte complexes/animal; four animals per treatment).

 
In addition to differences in cumulus expansion, differences were apparent in the extent to which the cumulus-oocyte complexes had detached from the mural granulosa cell wall in the unruptured follicles. Nine hours after hCG treatment, 88% of the complexes were detached from the mural granulosa cells in wild-type ovaries, whereas only 21% were detached in RIP140 null mice (Fig. 4BGo). In RIP140 heterozygous mice, which exhibit a partial anovulatory phenotype (22), 37% of the complexes were attached, and 63% were detached, independent of the diameter of the complex (Fig. 4BGo).

We next examined whether the failure to release oocytes could be explained simply by the observed (at 9 h) impaired detachment of the cumulus mass from the mural cells or whether there was an additional failure of the follicle wall to rupture. Examination of the ovaries at 12 h after hCG treatment showed that most of the follicles in the wild-type ovaries had ruptured and collapsed, but the ovaries from RIP140 null females at this time showed no evidence of follicular rupture, and the follicles retained large antra, some of which were blood filled. RIP140 heterozygous females, which have a reduced ovulation rate, showed a mixture of newly ruptured and unruptured follicles, some of which were hemorrhagic (data not shown). No sign of follicular rupture was evident in the RIP140 null ovaries even 15–18 h after hCG treatment. However, by this time the retained cumulus masses of RIP140 null and heterozygote mice were often sparse and poorly organized, with some oocytes being entirely denuded of any surrounding cumulus; few showed any obvious signs of attachment of the cumulus to follicle wall, but were retained in the unruptured follicles. These observations indicate that although RIP140 is not required for follicular growth or for oocytes to resume metaphase I of meiosis, it is necessary for both normal cumulus expansion and final follicular rupture.

RIP140 null ovaries have reduced expression of genes required for cumulus expansion
Cumulus expansion depends on the coordinated expression of a number of genes in response to the LH surge (Fig. 5AGo), and the array data show that the expression of nine genes involved in this process (listed in Table 1Go) is impaired by at least 2-fold in RIP140 null mice after hCG treatment. The expression of six of these genes was determined by quantitative PCR analysis of individual ovarian RNA samples from at least three wild-type and RIP140 null mice stimulated with gonadotropins. This analysis confirmed that the expressions of hyaluronan synthase-2 (HAS-2), TSG-6, amphiregulin, COX-2, chondroitin sulfate proteoglycan 2 (versican), and disintegrin and metalloproteinase with thrombospondin-like repeats-1 (ADAMTS-1) were all induced in wild-type mice after hCG treatment (Fig. 5BGo), whereas expression was markedly reduced in RIP140 null mice (Fig. 5BGo and Table 1Go).



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  		FIG. 5. Genes necessary for cumulus cell expansion are not induced in RIP140 null mice. A, Schematic representation of signaling pathways required for complete cumulus cell expansion. The genes in bold contribute directly to the structural integrity of the extracellular matrix (adapted from Ref. 5 ). B, RNA was isolated from the ovaries of immature wild-type (WT) and RIP140 null [knockout (KO)] mice after no treatment, 48 h after PMSG alone, or 3 h after hCG following PMSG. The expression of genes required for cumulus expansion was assessed using quantitative PCR. The expression level of each gene is normalized against the expression of L-19, and each bar represents the combined average expression of that gene for at least three animals, with the error bars representing the SEM for that group. Each group included all samples included in the array pool plus additional samples that had also responded to the hormone treatments. EGF, Epidermal growth factor; GDF-9, growth differentiation factor-9; PR, prostaglandin receptor.

 

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  		TABLE 1. Genes involved in the cumulus cell expansion are not induced in the RIP140 null mice in response to LH

 
We conclude that the reduced expression of structural and signaling molecules required for synthesis of the cumulus granulosa cell extracellular matrix combined with an up-regulation of several genes implicated in promoting strong cell-cell interactions contribute to the defect in cumulus expansion in RIP140 null mice and the failure of the follicles to rupture in response to LH.

Analysis of aberrantly regulated genes in distinct ovarian cell types
Although the above analyses provided an indication of differences in gene expression in the whole ovaries of wild-type and RIP140 null mice, the ovulatory defects in RIP140 null mice occur within the highly defined structural organization of the follicle. Therefore, to investigate the possible relationship between RIP140 and aberrantly regulated genes in relation to the discrete structural components of the follicle, we performed expression analysis on specific ovarian cell types isolated using laser capture microscopy. We focused on three groups of cells: cumulus-oocyte complexes, mural granulosa cells, and thecal/interstitial cells. The expressions of progesterone receptor, LH receptor, and RIP140 were used as markers for effective isolation of these cell populations (Fig. 6Go). Previous immunocytochemistry and in situ hybridization demonstrated that both receptors are induced in response to hormones: the LH receptor after PMSG treatment in mural granulosa and thecal cells, and the progesterone receptors after hCG treatment in mural granulosa cells (27, 28). Using laser capture microscopy, we found that the expressions of these two genes were markedly increased in the appropriate cell types after PMSG and hCG treatments, respectively, as predicted in both wild-type and RIP140 null ovaries (Fig. 6Go). RIP140 mRNA was detected in the mural granulosa cells of wild-type ovaries at all time points, but expression was increased in response to PMSG treatment and was decreased after hCG stimulation. We then studied the mRNA expression of some of the aberrantly expressed genes listed in Table 1Go and Fig. 1CGo in the same isolated cell populations. Although LH receptor expression was not dramatically altered in RIP140 null ovaries, the mRNA for amphiregulin in the mural granulosa cells, a downstream target of LH, was reduced in RIP140 null mice. This reduced expression is in accordance with microarray and quantitative PCR analyses of the whole ovary, but identifies granulosa cells as the site of expression (Fig. 6Go). Two genes involved in the formation of extracellular matrix were then studied. Cartilage acidic protein was undetectable in all three cell compartments derived from wild-type mice, but was expressed in both mural and cumulus granulosa cells regardless of the hormone status of RIP140 null cells (Fig. 6Go). Finally, HAS-2 mRNA was detected exclusively in cumulus-oocyte complexes in response to hCG, but this expression was reduced in the RIP140 null cumulus-oocyte complexes (Fig. 6Go). These observations are also in agreement with the microarray and quantitative PCR analyses.



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  		FIG. 6. Quantification of gene expression in distinct ovarian cell types. Ovaries of immature wild-type (WT) and RIP140 null [knockout (KO)] mice were removed after no treatment, 48 h after PMSG injection, or 3 h after hCG (given 48 h after PMSG). The ovaries were frozen and cryosectioned. Sections were briefly fixed and visualized with hematoxylin, and three cell populations were isolated using laser dissection microscopy. The cell populations isolated were mural granulosa cells (GR), cumulus granulosa cells (CU), and theca/interstitial cells (a population that may contain ovarian epithelium and vasculature; T/I). RNA was isolated from the cells and used for cDNA synthesis. As controls, the relative expression levels of RIP140, LH receptor, and progesterone receptor were assessed using real-time quantitative PCR. Then the expressions of three aberrantly regulated genes were assessed. The expression level shown is normalized against that of L-19 expression and represents a percentage of the highest value detected for each individual gene in three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The absence of RIP140 affects the expression of a large number of functionally distinct genes in both unstimulated and hormonally stimulated ovaries. This is consistent with a regulatory role for RIP140 in mediating nuclear receptor transcriptional control. We have previously reported that RIP140 shows a hormonally regulated spatial and temporal distribution within the follicle (19, 29), and the current study confirms and extends these observations. However, it is currently difficult to position RIP140 in specific ovarian signaling pathways that are each subject to control by other intra- and extraovarian factors. Thus, although the complete anovulatory phenotype of RIP140 null mice resembles that of progesterone receptor and COX-2 knockout mice (9, 12), the deficiency of RIP140 appears to affect a wider set of signaling pathways than either of these other genes.

The possibility cannot be excluded that the anovulatory phenotype ultimately reflects some of the early differences in gene expression seen in untreated and PMSG-treated RIP140 null mice. This could occur through inappropriate structural foundations or signaling pathways being established in the growing follicle before the LH/hCG surge. In this context, PMSG-stimulated follicles from RIP140 null animals showed higher expression levels of a variety of genes, including cartilage acidic protein, which was originally identified in cultured chondrocytes and predicted to be involved in the extracellular matrix (30); desmocollin 2, a cell adhesion molecule that is a component of desmosomes (31); syndecan 2, a cell adhesion molecule implicated in matrix formation, cell migration, and growth factor binding; and coronin actin-binding protein 1C, which is involved in cytokinesis, motility, and signal transduction (32). However, despite the range of genes aberrantly regulated, our observations suggest that follicular growth is morphologically normal and that the only obvious morphological ovarian phenotypes in RIP140 null mice are disturbed cumulus expansion and a failure of follicles to rupture in response to the preovulatory gonadotropin surge.

A number of studies have also demonstrated that the dramatic morphological and physiological changes associated with follicular growth and ovulation are reflected in complex changes in gene expression. Leo et al. (33) initially used RNA extracted from ovaries to show the potential of DNA arrays to identify genes showing induction or repression after the preovulatory LH surge in rats. However, such analyses of whole ovaries inevitably obscure the events occurring in individual cell types, and Jo et al. (34) established a rat ovarian gene database using granulosa cells and extrafollicular tissues as well as whole ovaries. This showed that the expression patterns of hundreds of genes are altered after PMSG and/or hCG treatments. Similarly, serial analysis of gene expression (SAGE) analysis of granulosa cells isolated from mouse preovulatory follicles showed that 216 genes were down-regulated 12 h after hCG treatment, whereas 499 were up-regulated (35). The complex interdependence of the various preovulatory pathways is also reflected in the observation that the absence of bikunin affects a range of genes encoding for stress-related, apoptosis-related, protease, signaling, and extracellular matrix molecules after hCG treatment in mice (36).

Remodeling of the extracellular matrices within the follicle and its surrounding tissues is integral to ovulation (37), with the expansion of the cumulus being one of the most obvious and biologically important events. The LH surge results in the coordinated induction of several signaling pathways (38) within the cumulus and mural granulosa cells, and these, in turn, initiate the expression of a variety of structural molecules, including the major matrix component, hyaluronan (14, 17, 39). Changes in the follicular basement membrane also occur that allow the influx of specific serum components, which then interact with the major structural molecules secreted within the follicle (40). The final cumulus cell extracellular scaffold includes the light and heavy chains of serum I{alpha}I (40), TSG-6 (41, 42), and versican (43, 44), all of which interact with hyaluronan (44, 45, 46, 47). This muco-elastic expanding matrix accumulates around the cumulus cells, causing them to disperse and ultimately to lose their close attachment with the mural granulosa cells, freeing the cumulus-oocyte complex for subsequent release from the ruptured follicle. Defective cumulus expansion is evident in mice null for these hyaluronan-binding factors; for example, bikunin (the light chain of I{alpha}I) (15) or TSG-6 (14). Versican is another hyaluronan-binding factor that is believed to be important in matrix stability and is normally induced by the preovulatory LH surge (43). The cleavage of versican by ADAMTS-1 is also vital for matrix formation and stability (43, 48). This is consistent with the observation that progesterone receptor null mice, which have reduced levels of the progesterone receptor target gene ADAMTS-1, have reduced levels of the versican cleavage product and are unable to ovulate. Similarly, pentraxin-3 is important for matrix stabilization once ovulation has occurred, and ovulated oocytes from mice lacking pentraxin-3 are usually denuded of cumulus cells.

Our own observations of the physical characteristics associated with the anovulatory phenotype of RIP140 null mice suggest that RIP140 may have a key regulatory role in the various pathways leading to extracellular remodeling in the preovulatory period. A schematic diagram of the signaling pathways leading to normal expansion and stabilization of the cumulus matrix is shown in Fig. 5AGo. We propose that defective expansion and poor stabilization of the cumulus matrix in RIP140-deficient mice reflect a multisite action of this nuclear receptor corepressor. The reduced induction of HAS-2 by LH/hCG in RIP140 null mice would account for the lower levels of hyaluronan observed. In turn, this may be accentuated by an increased expression of stabilin-2, a transmembrane hyaluronan binding protein implicated in the turnover of this matrix component (49). The decreased expression levels of versican, its cleavage protein ADAMTS-1, CD44 (a hyaluronan receptor), and TSG-6 observed in RIP140 null ovaries would lead to destabilization of the hyaluronan matrix skeleton. The levels of TSG-6 are controlled by COX-2, which, in turn, appears to be controlled by members of the epidermal growth factor ligand superfamily (e.g. amphiregulin, betacellulin, and epiregulin) (10), all of which are reduced in RIP140 null mice after hCG treatment. Although disruption of any one of these pathways might have been sufficient on its own to induce the same anovulatory phenotype, our studies indicate that RIP140 is involved in regulating all of these key components.

Although defective cumulus expansion is usually linked to significantly reduced ovulation rates in other mouse models, this is not an all or none dependency. Ovulation still occurs, albeit at reduced rates, in mice lacking TSG-6 (14), pentraxin 3 (16), growth differentiation factor-9 (50), bikunin (15, 51), phosphodicsterase 4D (10), and the prostaglandin E2 receptor (17). Therefore, the impaired cumulus expansion in RIP140 null mice may not be sufficient to explain the failure to ovulate. Tissue remodeling must also occur in basal membranes, thecal and interstitial layers, and the overlying ovarian surface at the apex of the follicle to allow the follicle to rupture (48, 52, 53, 54, 55), Vascular changes accompany these events, including increases in vascular permeability and vascular invasion of the ruptured follicle. Perifollicular smooth muscle contraction at the base of the follicle may also be essential for final rupture (56, 57). The mechanisms underlying all of these various changes at the periphery of the follicles and ovary are more poorly understood than those of cumulus expansion. They appear to involve a coordinated remodeling of the extracellular matrices via protease cascades involving members of the matrix metalloproteinases and ADAMTS families (48, 55, 58). Although we did not study gene expression any later than 3 h after hCG treatment, it is interesting to note that many of the up-regulated genes in RIP140 null mice, even when untreated or after PMSG stimulation, were those encoding for proteins known to be involved in cell-cell interactions, matrix attachment, or proteolytic processes (e.g. serpin A3N, stabilin-2, desmocollin-2, and cartilage acidic protein). It may be that an increased expression of such genes both during follicle growth and before ovulation results in a follicular structure inappropriate for ovulation.

The observations that the lack of RIP140 affects so many components of both cumulus expansion pathways and remodeling processes within 3 h of the hCG signal are dramatic. However, although the lack of RIP140 impairs some of these preovulatory signaling pathways, others remain functional. The integrity of these is evident from the fact that PMSG stimulated the initial up-regulation of the LH receptor, whereas hCG induced the expected up-regulation of the progesterone receptor as well as some induction of both amphiregulin and HAS-2 (albeit at much lower levels than in the wild-type animals). Morphologically, other apparently normal responses included germinal vesicle breakdown, luteinization, and vascular invasion of the luteinizing follicles. This distinction among the affected, partially affected, and unaffected pathways downstream from the LH surge combined with those alterations in gene expression before this point provides a focus for elucidating the precise role of RIP140 in regulating the events leading to ovulation.


   Acknowledgments
 
We thank Dr. R. Tammi for providing probes for the detection of hyaluronic acid, and S. Stubbs for assistance with sample preparation. We are also grateful to the staff of the Medical Research Council/Clinical Sciences Centre/Imperial College Microarray Center for invaluable assistance with the microarray hybridization, staining, and data analysis.


   Footnotes
 
This work was supported by Biotechnology and Biological Sciences Research Council Grants 01/B1/S/07260 and 28/S15893 (to J.M.A.T. and V.P.) and Wellcome Trust Grant 061930 (to J.H.S.).

First Published Online May 26, 2005

Abbreviations: ADAMTS-1, A disintegrin and metalloproteinase with thrombospondin-like repeats 1; COX-2, cyclooxygenase-2; HAS-2, hyaluronan synthase-2; hCG, human chorionic gonadotropin; I{alpha}I, inter-{alpha}-inhibitor; PMSG, pregnant mare serum gonadotropin; TSG-6, TNF-stimulated gene 6.

Received March 23, 2005.

Accepted for publication May 16, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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