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Endocrinology Vol. 144, No. 3 1020-1031
Copyright © 2003 by The Endocrine Society

ARTICLE

Hormone-Regulated Expression and Localization of Versican in the Rodent Ovary

Darryl L. Russell, Scott A. Ochsner, Minnie Hsieh, Sabine Mulders and Joanne S. Richards

Department of Molecular and Cellular Biology (D.L.R., S.A.O., M.H., J.S.R.), Baylor College of Medicine, Houston, Texas 77030; and Target Discovery Unit, N.V. Organon, 53 40 BH Oss, The Netherlands

Address all correspondence and requests for reprints to: JoAnne S. Richards, Ph.D., 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
 
During ovulation, production of a specialized hyaluronan (HA)-rich matrix cross-linked by associated HA binding factors causes expansion of the cumulus oocyte complex. Versican is a member of the hyalectan family that binds HA, provides structure and elasticity to tissues, and impacts cell motility and adhesion. In these studies, we sought to determine whether versican is synthesized by ovulating follicles and localizes along with HA in the expanded cumulus oocyte complex matrix in rodent ovaries and whether its expression and/or localization is altered in anovulatory mutant mice. Analysis of mRNA and protein identified isoforms V0, V1, and V3 versican in mouse and rat ovaries throughout follicular development. In situ hybridization localized versican mRNA most specifically to the granulosa cells. Expression was not significantly altered by estradiol or FSH treatment but was increased up to 10-fold during the periovulatory period after human chorionic gonadotropin treatment. In cultured granulosa cells, forskolin and phorbol 12 myristate 13-acetate or FSH + testosterone increased expression of versican. Immunohistochemical analyses verified versican protein in ovulating follicles localized to the expanded cumulus matrix as well as adjacent to the basement membrane. After ovulation, versican was localized around newly formed corpora lutea and vasculature. Unexpectedly, immunohistochemical analyses also demonstrated versican protein on granulosa cells in early primary and small antral follicles. Versican expression and localization were not altered in progesterone receptor or cyclooxygenase-2 null mice, suggesting that transcription of the versican gene is not a target of these two ovulatory mediators. These observations suggest that versican (V0, V1, and V3) is a matrix component of the granulosa layer throughout folliculogenesis and is enriched in remodeling matrices during ovulation and neovascularization of the corpora lutea.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OVULATION OF A MATURE oocyte and its accompanying mass of cumulus cells is essential for female reproductive success. This process is complex and involves specific integration of local signals from the oocyte and somatic cells of the ovary as well as endocrine signals from the pituitary. Ultimately, in response to the ovulatory LH surge, mucification or matrix expansion of the cumulus cell oocyte complex (COC) occurs. Regulated degradation of the follicle wall then allows rupture of the apical surface and release of the COC. For these events to occur, the LH surge initiates expression of critical genes in a precise temporal and spatial pattern (1). The products of some of these genes selectively impact the formation of the COC matrix, which is essential for extrusion of the oocyte. The cyclooxygenase-2 (COX-2) gene is induced in cumulus cells as well as mural granulosa cells within 2 h after the LH surge, and expression peaks within the follicle around 4 h (2, 3). After this time, COX-2 expression becomes restricted to the cumulus cells (4, 5). The resulting synthesis of prostaglandin E2 and its action via the prostaglandin E receptor type 2 receptor are essential for complete cumulus expansion and subsequent ovulation (6, 7). Induction of the hyaluronan synthase-2 (HAS-2) gene mediates the rapid local production of large hyaluronan (HA) molecules that form the obligatory backbone of the complex (8, 9, 10). HA is bound by hyaladherins including inter-{alpha}-trypsin inhibitor (I{alpha}I), a serum protein essential for ovulation (11, 12, 13, 14) and TNF{alpha}-stimulated gene 6 (TSG-6) (15, 16). Upon entering the ovulating follicle, the heavy chains of I{alpha}I are covalently cross-linked to HA, thus stabilizing the expanding cumulus matrix. TSG-6 is a hyaladherin produced by granulosa and cumulus cells and is induced after the LH surge (15). In addition to binding HA, TSG-6 is also reported to interact with I{alpha}I (16, 17) and with chondroitin-4-sulfate side chains of proteoglycans, such as versican (18, 19). The exact composition and function of COC matrix complexes remain to be clearly defined; however, recent observations suggest that HA and versican share functional links during morphogenesis and cancer (20, 21). Thus, versican and HA may be coexpressed and critical for COC matrix formation/function.

Versican (proteoglycan M) is a proteoglycan hyalectan with a broad tissue expression profile (22). In addition to an N-terminal link-module HA binding domain, versican possesses a C-terminal lectin-like domain that may bind cell surface or matrix molecules. Dual epidermal growth factor (EGF)-like modules that can stimulate cell proliferation as well as a complement regulatory like protein domain complete the C terminus (Fig. 1Go). Many functional properties of the protein are determined by two glycosaminoglycan (GAG) attachment domains that are modified to carry long chondroitin sulfate side chains, giving the protein a large molecular size (up to 1600 kDa), strong negative charge, and hydrodynamic properties. Alternative splicing events of a single gene (23, 24, 25) generate either a full-length protein possessing both GAG attachment domains (V0), the ßGAG domain alone (V1), {alpha}GAG alone (V2), or neither GAG domain (V3). Among its reported activities, versican may confer structural integrity to tissues and antiadhesive promigratory effects on vasculature (26) that may be antagonized by the V3 variant (27). Mice with a versican null gene mutation die during embryogenesis due to defects in the developing heart (21), an identical phenotype to that observed in mice null for HAS-2 (20). These observations suggest a close functional link between these two genes and demonstrate the important role of each in tissue morphogenesis. A dermatan-sulfated proteoglycan of large size has been reported in COC and suggested to be versican (28). Chondroitin sulfated proteoglycans nidogen (entactin) and brevican, as well as versican, were identified in the basement membranes of bovine ovarian follicles (29) and in human follicular fluid (30). Thus, there may be important structural roles for such proteoglycans in the follicle matrix. Additionally, the accumulation of a versican- and HA-enriched matrix in breast and ovarian tumors is a strong negative predictor of patient survival (see Ref. 31 for review).



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  		Figure 1. Domain organization of versican isoforms and primer design for isoform specific amplification. Domains of the versican protein backbone include signal peptide (sig), HA binding (HA), GAG attachment domains {alpha} and ß, EGF, lectin-like (L), complement regulatory-like (C), and tail (T) regions. Alternative splicing of the full length V0 mRNA produces four unique variants that were identified using RT-PCR primer sets indicated by arrows that span domain interfaces specific to each isoform. The Vers 0/1 primer set detects V0 and V1 isoforms with equal efficiency. The product of this PCR was subcloned and used in in situ hybridization analysis.

 
Ovulating follicles and surrounding stroma express and control a range of active proteases that specifically degrade the follicle/ovary wall. Few mutant mouse models with specific defects in follicular rupture exist; however, one such model is the progesterone receptor (PR) knockout (PRKO) mouse. These mice form corpora lutea (CL) with entrapped expanded COCs (31, 32). Two proteases that are expressed abnormally in PRKO mice that could account for the failure of follicle rupture are Cathepsin L and a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS)-1. The essential substrates for these proteases in the ovulatory follicle are not yet known. Cathepsin L is a broad spectrum protease that may degrade extracellular matrix (33, 34). Importantly, ADAMTS-1 has been shown to degrade the proteoglycans aggrecan (35) and versican (36). Versican is cleaved by ADAMTS-1 within the ßGAG domain present in V0 or V1 isoforms. Thus, versican may be a target of ADAMTS-1 in the ovulating follicle.

Versican has the potential to mediate several of the functional properties of the ovulating follicle. These include, but are not limited to, binding HA and cross-linking of the expanding cumulus matrix, contributing to the structural matrix of the follicle wall, or promoting cell detachment and migration during postovulatory wound healing or vascularization of the corpus luteum (CL). We therefore sought to identify the isoform-specific regulation of versican in the ovulating ovaries of mice and rats. Versican in other tissues such as cartilage and aorta localizes to highly elastic matrices. Therefore, we also examined whether versican protein localized to the viscoelastic COC matrix in periovultory ovaries. The expression and localization of versican were further investigated in anovulatory PRKO as well as COX-2 null mice. These studies yield new insights into the composition and expression of proteoglycans during follicular growth and the complexity of the expanding cumulus matrix during the ovulatory process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Pregnant mare serum gonadotropin (PMSG) (Gestyl) was purchased from Professional Compounding Center of America (Houston, TX). Human chorionic gonadotropin (hCG) (Pregnyl) was purchased from Organon Special Chemicals (West Orange, NJ). Forskolin (Fo) was purchased from Calbiochem (San Diego, CA). Phorbol myristate acetate (PMA) and testosterone (T) were purchased from Sigma (St. Louis, MO). Ovine FSH-16 was a gift from the National Hormone and Pituitary Program (Rockville, MD). Reverse transcriptase and Taq polymerase were from Promega Corp. (Madison, WI). Oligonucleotides were synthesized by Sigma-Genosys (Houston, TX). Rabbit anti-versican antibody (Vc) raised against recombinant human versican was generously provided by Dr. Richard LeBaron (University of Texas San Antonio).

Animals and hormone treatments
Wild-type C57BL/6 mice were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN). COX-2 null mice (37) were obtained from The Jackson Laboratory (Bar Harbor, ME). PRKO mice were obtained from Dr. John P. Lydon (38, 39). Follicular growth and ovulation were stimulated in mice at 21 d of age by the following hormonal regimen: 4 IU PMSG was injected ip followed 48 h later with 5 IU ip injected hCG. Ovaries were isolated from untreated, PMSG-treated, or PMSG and hCG-treated mice at selected time intervals as indicated in Results and figure legends. In some experiments expanded COC were isolated from PMSG + hCG 12-h-stimulated ovaries by selective puncture of preovulatory follicles with a fine 27 -gauge needle. After collection of all expanded COC, the remaining granulosa cells were harvested by further repeated puncture of follicles.

Immature female Holtzman Sprague Dawley rats (23–25 d of age) used for granulosa cell isolation and culture were purchased from Harlan Sprague Dawley, Inc.

Hypophysectomized (H) rats were purchased from Harlan. Ovarian follicle growth and differentiation were stimulated in H rats by exogenous hormonal treatment as described previously (40, 41). Commencing 3–4 d after H, rats received daily sc injections of 17-ß estradiol (1.5 mg) for a consecutive 3 d (HE), following which rats were treated for 2 d by sc injection of 1 µg FSH twice daily (HEF 48 h). Ovulation was induced in HEF rats by tail vein injection of 10 IU hCG (HEF/hCG 4–48 h). After each hormone treatment for the times indicated, groups of three to four animals were euthanized, ovaries extirpated, and granulosa cells were isolated by ovarian puncture for preparation of RNA. Whole ovaries were also fixed in 4% paraformaldehyde, paraffin embedded after dehydration, and sectioned for in situ hybridization and immunohistochemistry as described below.

All animals were provided food and water ad libitum and housed under a 12-h light, 12-h dark schedule and treated in accordance with the NIH Guide for Care and Use of Laboratory Animals. Protocols were approved by the Institutional Animal Care and Use Committee, Baylor College of Medicine (Houston, TX).

RNA isolation from whole mouse ovaries and isolated rat granulosa cells
Ovarian RNA was obtained by tissue homogenization in TRIzol Reagent (Life Technologies, Inc., Gaithersburg, MD) followed by RNA precipitation in isopropanol. Recovered RNA was then washed in 70% ethanol and dissolved in ribonuclease-free water. RNA concentration was determined and samples were stored at -80 C until use.

Granulosa cells from rat ovaries stimulated with PMSG (10 IU) for 48 h or estradiol for 72 h were isolated and cultured as previously described with modifications (42). Briefly, cells were cultured overnight in DMEM:Ham’s F-12, 5% fetal bovine serum, 100 IU/ml penicillin, and streptomycin at a density of approximately 1 x 106 cells per well in a six-well plate (Falcon, BD Biosciences, Lexington, KY). Cells were then washed and cultured in serum-free medium the absence or presence of indicated agonists. Cells were stimulated with Fo (10 µM) plus PMA (20 nM) or FSH (50 ng/ml) plus T (10 ng/ml) for 2–48 h as indicated. Some cultures were treated with FSH/T for 48 h followed by Fo and PMA for an additional 2 h. Granulosa cells were harvested in TRIzol reagent and RNA was prepared as described above.

RT-PCR analysis
RT-PCR primers were generated using a web-based primer prediction algorithm (43). Versican primer pairs that spanned exon boundaries unique to each isoform were designed based on mouse sequence described for versican V0 (accession no. D28599). In addition, a primer set that amplified both V0 and V1 isoforms with equal efficiency were designed to enable quantitative analysis of the total versican production (Table 1Go). Mouse ribosomal L19 primer pairs were based on mouse rpL19sequence (accession no. M62952) (Table 1Go). The same primer sets were also employed for studies of rat tissues.


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  		Table 1. Versican variant specific PCR primer sequences

 
Briefly, RT-PCR was performed using 300 ng of total RNA reverse transcribed using 1x Thermocycle buffer, 250 ng oligo-deoxythymidine primer, 1 mM deoxy-NTPs, 4 mM MgCl2, 20 U RNAsin, and 2.5 U avian myeloblastosis virus-reverse transcriptase (RT) at 42 C for 90 min. The V0 and V1 specific isoforms were reverse transcribed under the same conditions; however, the specific reverse (3') primers were used to initiate the RT reaction because the region to which PCR primers were designed was up to 5 kb away from the mRNA poly-A tail. All other reactions including V2 and V3 employed poly-deoxythymidine RT-reaction primers. After completion of the RT reactions, the following were added: 500 ng of each primer, 2 µCi [32P]deoxy-CTP (ICN Radiochemicals, Los Angeles, CA), and 2.5 U Taq polymerase in 1x Thermocycle buffer and 2.5 mM MgCl2. PCR conditions were 94 C for 2 min followed by 25 cycles of 94 C for 30 sec, 60 C for 45 sec, and 72 C for 60 sec with a 72 C extension for 10 min (44). Primers for the ribosomal gene L19 were included as an internal amplification control in all except V0 and V1 specific PCRs. PCR products were separated on a 5% polyacrylamide gel and exposed to film. Products were quantified using a PhosphorImager and ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).

In situ hybridization
In situ hybridization was done as described by Wilkensen (45). Briefly, 35S-uridine triphosphate (Amersham Pharmacia Biotech, Piscataway, NJ)-labeled antisense and sense probes of mouse versican cDNA were made using the Riboprobe In-Vitro Transcription Systems kit (Promega Corp.). The TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA) was used to subclone the 406-bp RT-PCR product of the Vers 0/1 primer set generating a probe with general specificity for V0/1. Ovaries were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 7 µm onto silane-coated slides. After deparaffinization and rehydration, sections were pretreated with 20 µg/ml proteinase K and 0.1 mM triethanolamine/acetic anhydride before coating with labeled probe and incubated overnight at 55 C. Slides were washed with increasing stringency using 5x sodium chloride/sodium citrate (SSC) followed by 50% formamide/2x SSC/100 mM ß-mercaptoethanol for 30 min each at 65 C. Slides were then treated with 20 µg/ml ribonuclease A for 30 min at 37 C followed by final washes of 2x SSC and 0.1x SSC for 15 min at 65 C and dehydrated. To visualize 35S-uridine triphosphate label, slides were dipped in photographic NTB-2 emulsion (Kodak, Rochester, NY) and exposed for 3 d. D-19 developer and fixer (Kodak) were used to develop the slides followed by hematoxylin counterstain.

Immunohistochemistry
Tissue and cellular localization of versican was analyzed by immunostaining of 4% paraformaldehyde-fixed paraffin sections of ovaries of mice and rats. Rehydrated sections were treated for 10 min with 1 µg/ml proteinase K followed by chondroitinase ABC (Sigma), endogenous peroxidase activity was quenched by 10 min treatment with 0.1% H2O2 followed by PBS wash. Nonspecific antibody binding was blocked by 1 h incubation with 10% normal goat serum, following which Vc (1/200) in 10% goat serum was incubated with sections overnight at room temperature. After washing with PBS containing 0.025% Tween-20, biotinylated goat antirabbit antiserum (Vector Laboratories, Inc., Burlingame, CA) was added for 30 min, slides were washed, and streptavidin-conjugated horse radish peroxidase was applied for 30 min. Sections were incubated with 3,3'diaminobenzidine substrate (Vector Laboratories, Inc.) for 2 min, then dehydrated and mounted without counterstaining.

Western blot
Whole mouse ovary or isolated COC extracts were prepared by homogenization in 6 M urea 0.1% triton buffer containing protease inhibitors EDTA, benzamidine, phenylmethanesulfonyl fluoride, and aprotinin. Following extraction, 10-µg samples were diluted 1/10 in 0.08 M Tris acetate (pH 6.0) containing 0.02 U/ml chondroitinase-ABC (Sigma) and incubated for 1 h at 37 C. Samples were then resolved on 4–15% gradient acrylamide gels under reducing SDS-PAGE conditions and transferred to polyvinylidene difluoride membrane (Immobilon-P, Millipore Corp., Bedford, MA). Membranes were blocked by 1 h room temperature incubation with 3% nonfat milk, followed by 1-h incubation with Vc primary antibody 1/500 in 3% milk and washing in TBST (10 mM Tris, pH 7.5; 150 mM NaCl; and 0.05% Tween 20). Blots were then incubated with 1/10,000 dilution of horseradish peroxidase linked antirabbit IgG (Amersham Pharmacia Biotech), then washed with TBST. Enhanced chemiluminescence detection was performed using Pierce Chemical Co. SuperSignal according to the manufacturer’s specifications.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Versican mRNA is induced by LH/hCG during the periovulatory period
To study the expression of versican mRNA in the rodent ovary in detail, primers were designed to specifically detect each splice variant of this proteoglycan. Amplification of each variant was analyzed using RNA from whole ovaries of immature mice either untreated or after treatment with PMSG and hCG. Isoform specific RT-PCR analysis detected predominantly V0 and V1 splice products of the versican gene (Fig. 2AGo). These two isoforms each contain the ßGAG attachment domain, whereas V0 also contains the {alpha}GAG domain. The V3 variant that lacks both GAG attachment domains was also identified using primers to the conjoined HA-binding and EGF modules specific to this isoform. The expression pattern of each isoform in response to hormonal stimulation was similar, being strongly (>10-fold) up-regulated 4–12 h after hCG treatment. Specific RT-PCR analysis for the V2 isoform did not detect this variant in the mouse ovary, although the V2 primer set did detect a product of the expected size for V0 spanning the ßGAG domain (not shown), demonstrating that the primers were effective.



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  		Figure 2. Versican V0, V1, and V3 variants are expressed in mouse ovaries and induced by ovulatory hCG. A, Autoradiographs of 32P-labeled products generated by RT-PCR using specific primer sets for versican V0, V1, and V3 (see Table 1Go). Whole mouse ovary RNA was isolated from untreated immature (U), or preovulatory ovaries after treatment with PMSG (48 h) (P) or PMSG followed by an ovulatory dose of hCG for the time periods indicated. RT reactions were performed using the specific reverse primers for V0 and V1 or poly-deoxythymidine primers for V3 and L19. Specific RT-PCR amplification of versican V2 failed to detect any of this isoform in the ovary. All PCRs were performed under identical conditions, and identical exposure times are shown for each except V3, which was exposed 5 times longer. B, Semiquantitative RT-PCR analysis of the relative expression of V0/1 (upper panel) in comparison to L19 internal control coamplified in the same tubes (lower panel). PhosphorImager quantitation of relative versican expression compared with L19 from three independent experiments (mean ± SD) is shown in the histogram, and values above each bar indicate the fold induction observed over untreated samples.

 
There is little known evidence that V0 and V1 isoforms have different functions and in most cases both are coexpressed in versican-expressing tissues (22). Therefore, additional analyses of versican regulation by hormones were performed using a primer set common to V0 and V1 and using poly-deoxythymidine-primed RT reactions and coamplification of the L19 internal control. As with isoformspecific primer sets, low but detectable levels of versican mRNA were present in total RNA prepared from ovaries of nonhormone-primed mice or in mice treated with PMSG for 48 h to stimulate the growth of preovulatory follicles. However, within 2 h after hCG injection, versican mRNA was increased (Fig. 2BGo). Between 2 and 4 h after hCG treatment, versican mRNA expression increased up to 10-fold over that measured in preovulatory (PMSG treated) follicles. Versican message levels were maximal after 4–12 h of hCG treatment, a time nearing ovulation. At this time large periovulatory follicles with expanded COCs are present. Sixteen hours after hCG administration ovulation has occurred and is coincident with a decrease in versican mRNA that persisted during the postovulatory luteinization period.

To more fully investigate the tissue specificity and the action of specific hormones in versican regulation, RNA was extracted specifically from granulosa cells of H rat ovaries. Versican mRNA was low but detectable in H rat granulosa cells and remained low after treatment with estradiol (HE). Versican mRNA expression increased in response to FSH 48-h treatment (HEF) and more dramatically after hCG treatment (Fig. 3AGo). In RNA from whole ovaries 24 or 48 h after hCG treatment when large CL predominate the ovarian tissue mass, versican remained detectable. Versican message also increased but to a lesser extent in the hCG-stimulated residual ovarian compartment that is comprised of interstitial cells as well as granulosa cells of small follicles that are not released during the granulosa cells isolation procedure. Thus versican appears to be a major product of granulosa cells of ovulating follicles. To confirm this, the cDNA product amplified by primers Vers3' and Vers5' was subcloned into the pCR4-topo vector and used to generate sense and antisense riboprobes for in situ hybridization. Sections of rat (not shown) and mouse ovaries hybridized to the antisense probes showed specific granulosa cell expression of versican V0/1 mRNA after PMSG/hCG treatment (Fig. 3BGo). As in RT-PCR analyses the expression of versican mRNA was low in immature untreated or PMSG-treated mice and expression appeared maximal in granulosa cells of PMSG/hCG 4-h- and 12-h-treated ovaries. Versican message was predominantly localized to the mural granulosa cells but was also detected in the cumulus cells surrounding the oocyte.



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  		Figure 3. Versican V0 and V1 expression is specific to granulosa cells of mouse and rat ovaries. A, Autoradiograph and phosphorimage analysis of 32P-labeled RT-PCR using Vers 0/1 primers and total RNA extracted from isolated granulosa cells and residual ovaries of intact immature rats or H rats stimulated sequentially with estradiol for 3 d (HE), FSH for 48 h (HEF), and hCG for 12, 24, or 48 h. At 24 and 48 h, whole ovary RNA was used (WO). B, In situ hybridization of V0/1 expression in sections of wild-type C57Bl/6 mouse ovaries treated with PMSG (P) followed by hCG for the indicated times. Upper panels, Bright-field micrographs of hemaytoxylin-stained sections. Lower panels, Liquid emulsion autoradiographs of labeled antisense probe hybridization and sense control probe.

 
Signaling mechanism of versican induction in vitro
The intracellular signaling mechanisms that mediate induction of versican mRNA were investigated in cultured rat granulosa cells. Immature granulosa cells of preantral follicles isolated from E-treated rats or matured cells from PMSG-treated rats were placed in culture overnight then stimulated with specific agonists and total RNA subjected to RT-PCR analysis (Fig. 4Go, A and B, respectively). In nontreated immature and mature cells (control), levels of versican mRNA were readily detectable (Fig. 4Go). Immature cells treated in culture with FSH and T (FSH + T) or Fo, which activates adenylate cyclase to increase intracellular cAMP, along with phorbol ester PMA for 2, 4, or 12 h showed no change in versican expression. After 24 and 48 h of FSH + T treatment, versican mRNA increased 2-fold, and these in vitro differentiated cells acquired the ability to respond to Fo + PMA 2-h treatment (Fig. 4AGo). Likewise, mature granulosa cells from PMSG-treated rats responded within 2 h of Fo ± PMA or FSH + T treatment with increased versican expression (Fig. 4BGo). Longer treatments (4–48 h) similarly increased versican mRNA expression. However, cells differentiated in response to FSH + T 48-h treatment showed no additional response after addition of Fo + PMA. Thus, versican induction by LH-agonists Fo + PMA or FSH + T was dependent on differentiation of cells to preovulatory phenotype mediated by FSH + T maturation of immature cells in vitro or PMSG in vivo. Induction of versican mRNA in vitro was not as great as in vivo, suggesting that transactivation of versican expression after hCG treatment requires one or more signaling mechanisms not activated by FSH + T and/or Fo + PMA in vitro.



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  		Figure 4. Versican V0/V1 expression is activated by Fo, PMA or FSH and T in differentiated rat granulosa. Isolated granulosa cells from immature E-treated (A) or preovulatory PMSG-treated (B) rats were placed in culture and either untreated (control) or stimulated with agonists Fo to stimulate intracellular cAMP production and the phorbol ester PMA, to activate DAG-mediated signaling. Hormones FSH and T (FSH/T) were added to cultures alone or after pretreatment for 2 h with protein kinase A inhibitor H89 where indicated. Additional cultured cells were first induced to differentiate by treatment with FSH and T for 48 h then treated with or without Fo + PMA. Extracted RNA was subjected to RT-PCR analysis using the common V0/V1 primer set. Insets show representative autoradiographs of labeled V0/V1 and coamplified L19 products after 4% PAGE. Histograms show mean induction ± SD of V0/V1 mRNA compared with L19 internal controls. Data shown in histograms are from three repeated experiments.

 
Versican mRNA expression in anovulatory mutant mouse models
The temporal and spatial induction of versican mRNA is closely correlated with the periovulatory induction of several genes known to be critical for ovulation, most notably COX-2 (6, 37) and PR (38, 39). We therefore sought to determine whether the expression of versican was altered in mutant mice null for COX-2 or PR. In situ hybridization analyses showed that versican expression patterns in COX-2 null and heterozygous littermates (data not shown) were similar to that observed in wild-type C57Bl/6 (Fig. 3BGo). Namely, expression was limited to mural granulosa cells and cumulus cells of large preovulatory follicles from mice treated with PMSG and hCG. By semiquantitative RT-PCR analysis, the relative expression of V0/1 in COX-2 heterozygous and null littermates was found to be equivalent when analyzed either at 4 h or 12 h after hCG stimulation (Fig. 5AGo). Regulation of versican mRNA was also examined in mouse ovarian RNA from PR heterozygous and PRKO mice after PMSG or PMSG + hCG 4, 8, or 12 h treatment. Each treatment group included RNA from three individual mice that were analyzed together. Versican mRNA levels were low in both immature and PMSG-treated PR heterozygous and PRKO mouse ovaries (Fig. 5BGo). In contrast versican message increased markedly in PR+/- and PRKO ovaries, following stimulation with hCG. Student’s t test analysis determined that no significant difference existed in versican message levels between COX-2+/- and COX-2-/- after PMSG + hCG 12-h treatment. Likewise, versican mRNA levels were similar in PR+/- and PRKO mouse ovaries, although levels in hCG 8-h-treated ovaries were consistently but not significantly lower.



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  		Figure 5. Versican is expressed normally in anovulatory PRKO and COX-2-/- mouse ovaries. RT-PCR analysis of versican V0/1 expression in normally ovulating COX-2 or PR heterozygous mice compared with their anovulatory littermates with homozygous null mutations for COX-2 (A) or PR (B). RT-PCR was performed on RNA from COX-2-/- and normal COX-2+/- littermate ovaries after PMSG and hCG treatment for 4 h (n = 2) or 12 h (n = 4). RNA from three individual PR+/- or PRKO ovaries after PMSG or PMSG + hCG treatment for the indicated times were analyzed separately. Data presented are mean ± SEM of phosphorimage quantitated V0/1 expression normalized to L-19 amplified in the same tube. Student’s t test statistical analysis confirmed that versican expression was not significantly different in null vs. heterozygous littermates in each treatment group.

 
Protein expression and localization of versican in ovulating ovaries
Versican is a secreted matrix binding proteoglycan and therefore may act in different tissue compartments from that in which it is expressed. To determine the localization of versican protein, we obtained an antibody raised against human recombinant versican from Dr. Richard LeBaron (46). Mouse and rat ovarian sections were analyzed with this antibody by immunohistochemistry (Fig. 6Go). Initially, specific staining for versican was identified by comparison of adjacent sections of HEF + hCG 12-h-treated rat ovaries incubated with Vc or the same dilution of normal rabbit serum (NRS) and counterstained with hematoxylin to aid in the identification of tissue compartments (Fig. 6AGo). Distinct specific staining was detected in the expanded COC, granulosa cells, and theca layer of these periovulatory ovaries. Subsequent immunostaining was performed without counterstain to avoid masking of the signal. In the H rat ovary, versican was detected in the granulosa layer of primary follicles as early as stage 3 of growth and localized to the granulosa cell surfaces (Fig. 6AGo, black arrowheads). This even pattern of granulosa cell staining as well as staining in the theca cell layers (open arrowheads) was consistent through stage 7 (small antral) of follicle growth, the predominant follicle stages seen in H- and HE-treated rats. The level of detectable versican in the granulosa layer declined in large antral or preovulatory follicles, such as in the PMSG-treated mouse (not shown) or HEF rat ovary, whereas theca cell staining persisted (Fig. 6AGo). The strongest staining was detected in ovaries after hCG stimulation for 4–12 h (Fig. 6AGo, black arrows). In these ovaries, immunoreactive versican was localized predominantly in the antrum of ovulating follicles. Staining was clearly seen in the expanded matrix between cumulus cells and was much less abundant in the mural granulosa cells. The forming CL of ovaries 24 h after hCG treatment retained notable versican immunoreactivity specifically at the boundry of the CL (*). The sites of immunolocalized versican when compared with in situ hybridization of mRNA suggest that versican is produced and secreted by mural granulosa cells, diffuses and binds to matrix components of both the COC and theca interna. Occasional blood vessels in the ovarian stroma (not shown) and within CL were also immunopositive for versican.




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  		Figure 6. Versican protein is localized in multiple follicular compartments during hormonal stimulation of H rats, and localized normally in COX-2-/- and PRKO mouse ovaries. A, Sections of ovaries from H rats stimulated with estradiol (E), FSH (F), and hCG (HEF + hCG) as described for Fig. 3AGo were subjected to immunolocalization using the Vc antibody raised against human recombinant versican. Antibody binding was elucidated using HRP-label and 3,3'-diaminobenzidine staining (brown color) and sections were photographed at x20 (upper panels) and x63 (lower panels) magnification. Serial sections incubated with Vc antibody (1/500), or the same dilution of NRS and counterstained with hemaytoxylin are presented in the first two panels. Note immunopositive (brown) staining to the theca cell layers (tc), granulosa cells (gc), and COC (coc). Specific versican immunostaining was detected on the surface of granulosa cells in small growing follicles (black arrowheads). In large preantral follicles, and all antral follicles versican was localized in the theca cell layers (open arrowheads). Staining was diffuse in the granulosa cell layers of preovulatory follicles (HEF) but became intense within the follicular antrum within 2 h after administration of hCG. After 8–12 h, staining was intense in the expanded COC matrix (black arrows) with a fibrous appearance. In newly formed CL, versican staining persisted intensely in the region surrounding the CL structure (*) as well as in blood vessels within CL. B, Versican immunolocalization in mouse ovary sections from COX-2-/- or heterozygous littermates after PMSG + hCG 12 h or 16 h stimulation. COX-2-/- mice failed to ovulate (entrapped COC present after 16 h hCG) and COC expansion was delayed; however, staining was apparent within COC of COX-2-/- ovaries. C, Versican immunolocalization in littermate PR+/- or PRKO mouse ovaries after PMSG + hCG 12 h or 16 h of stimulation. PRKO mice also failed to ovulate (entrapped COC after 16 h), but versican immunolocalization and COC expansion were normal. D, Versican immunoreactivity remained associated with cumulus cell surfaces in COC present in the fallopian tube of WT mice after ovulation was induced with PMSG + hCG (16 h). Lower panels, Immunohistochemical analysis of sections from the same untreated mouse ovary incubated with NRS (1/500) or Vc (1/500) then counterstained with hematoxylin as in A. coc, COC; gc, granulosa cells; tc, theca cells. Examples of specific staining are indicated in primary follicles by black arrowheads, in theca cell layers by open arrowheads, and in COC by black arrows.

 
To further investigate this localization to expanded cumulus matrix we analyzed sections of anovulatory COX-2-/- mice. Impaired COC expansion in these mice is presumed to result from deficient production or incorporation of necessary components of the HA based matrix. Because versican mRNA was normally expressed in PMSG + hCG-treated COX-2-/- mice, we investigated the pattern of localization of the protein (Fig. 6BGo). Although COC expansion was impaired in COX-2-/- ovaries, the localization of versican to the cumulus cells and their surrounding matrix had a similar appearance to that of heterozygous controls.

In PRKO mice, preovulatory follicles fail to rupture presumably due to a defect in the matrix remodeling required to breach the follicle/ovarian wall. Versican is a known substrate for the protease enzyme ADAMTS-1 that is a downstream target of PR action. We therefore compared versican immunolocalization in PRKO ovaries to normal PR+/- littermate controls. After PMSG + hCG treatment for 12 h, the relative level and localization of versican protein was not altered in PRKO ovaries (Fig. 6CGo). These results, however, do not determine whether versican has been cleaved by ADAMTS-1 or if its function is altered.

Versican immunoreactivity remained associated with the surface of ovulated cumulus cells present in the oviduct of PMSG + hCG 16 h-treated normal mice (Fig. 6DGo). However, staining of the extracellular matrix surrounding oocytes became diffuse after ovulation.

Western blot with the Vc antibody using protein extracted from whole mouse ovaries either unstimulated or treated with PMSG for 48 h or PMSG followed by hCG for 12 or 24 h identified two major bands of immunoreactive versican after chondroitinase ABC digestion of samples. Bands of immunoreactivity of approximately 400–500 kDa, the expected size V1 and V0 versican, were faintly detected in untreated and PMSG-treated mouse ovaries, and each was strongly increased after hCG (Fig. 7Go). Thus, as seen in mRNA analyses, both ßGAG-containing isoforms of versican protein are present and detectable in ovulating mouse ovaries. In a second experiment, Western blot analysis of versican was performed on expanded COC or mural granulosa cells isolated from PMSG + hCG 12-h-treated mouse ovaries. Four hundred to 500 kDa of versican was detectable in both compartments but was far more abundant in COC. Additionally, a 70-kDa band of immunoreactivity was clearly detectable in COC. This most likely represents the degradation product of V1 versican, through the action of a hyalectanase enzyme such as ADAMTS-1 (36).



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  		Figure 7. Versican protein in whole ovaries and isolated granulosa cells and cumulus oocyte complexes of periovulatory follicles. Western blot of versican protein expression in wild-type C57Bl/6 mouse ovaries either unstimulated (U), PMSG treated, or PMSG + hCG treated for 12 or 24 h. Each lane contains 10 µg of protein from whole ovary extracts of individual mice. Very large (400–500 kDa) bands of versican immunoreactivity were detected in samples at all stages of follicular development. Right panel, Versican Western analysis of 10 µg protein extract from isolated granulosa cells or COC pooled from 5 PMSG + hCG 12 h-treated mice. Versican immunoreactivity was present in both follicular compartments but more concentrated in COC. A smaller approximately 70-kDa band of versican immunoreactivity was also detected in COC.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
HA production and stabilization have been shown to be essential for successful ovulation. Here we document for the first time that versican, an HA binding protein, is expressed and hormonally regulated in ovulating follicles of mice and rats. The mRNA for both the V0 and V1 isoforms of versican that contain {alpha} + ß or ßGAG attachment domains, respectively, and the GAG-deficient variant V3 were detected at all stages of folliculogenesis. The V2 isoform that is mostly associated with neuronal tissues (20) was not detected. Each variant detected was strongly induced in mural granulosa cells by an ovulatory dose of hCG. Likewise, versican protein was detected in granulosa cells of primary and growing follicles but was most intensely detected within expanded COC matrix of ovulatory follicles.

During the periovulatory period, follicles respond to the LH surge by expressing genes required for ovulation and undergo the necessary changes to facilitate rupture and release of the COC. Inducible factors involved in ovulation include COX-2 (2, 4), TSG-6 (15, 16, 47), PR (39, 48), ADAMTS-1 (31, 49), Egr-1 (50, 51), and CCAAT/enhancer binding protein-ß (52). In particular, COX-2 and PR are confirmed essential ovulatory genes. The coordinate increase in versican expression within the periovulatory period suggests that it may also play one or more roles in one or more ovulatory processes. To this end, we analyzed versican expression in PRKO and COX-2-/- mice. No alteration in the LH/hCG-mediated induction was observed; thus, it can be concluded that the anovulatory phenotypes of these mice are not related to changes in versican expression. However, the functional activity of versican may depend on proteolytic cleavage and could be altered because versican has been shown to be a substrate for the protease ADAMTS-1 (36), which is deficient in the PRKO mice (31) as discussed below.

Among LH surge-induced genes, the mechanisms controlling activation of the proteoglycan-M gene encoding versican is somewhat unique. Maximal induction in vivo occurred from 4–12 h, rather than the more transient expression observed for other LH-induced genes mentioned above (1). Furthermore, cultured granulosa cells from immature rats were induced to express increased levels of versican by Fo + PMA as well as FSH + T only when differentiated to the preovulatory stage. Relatively little is known about the regulation of versican mRNA. Within the -632 bp of 5'-promoter sequence that have been analyzed for the human versican gene, binding sites for AP2 as well as cAMP response element binding protein, CCAAT/enhancer binding protein-ß, and Sp1 were identified (53). Each of these transcription regulators are functionally important in granulosa cells and could potentially mediate the effect of LH on versican expression. In the ovary, versican appears to be regulated by diverse mechanisms, those that impact expression early in follicular development and those that induce expression in ovulating follicles. In vascular smooth muscle cells, versican is induced by platelet-derived growth factor (54) and TGFß (55, 56, 57). The expression of versican in primary growing follicles before the FSH-responsive growth stage might be mediated by a TGFß family member such as activin (58) or growth and differentiation factor-9 (4), which is also necessary for periovulatory expression of COX-2. Although the LH surge is the initiator signal for maximal expression in preovulatory follicles, the downstream mediator(s) of versican induction at this time are not yet known. Intriguingly, expression of versican is reduced in TAFII105 null mice in which follicular growth is severely altered (59).

Interestingly, versican protein was localized in follicular compartments distinct from the mural granulosa cells that most highly express the mRNA. Our detection of versican in the COC matrix is in agreement with previous reported detection in human follicular fluid (30) as well as in mouse COC expanded in vitro (28). The presence of versican as well as HA in the expanded COC matrix suggests that the protein may play a role in the organization or stabilization of the COC matrix. This localization to expanded COC also closely correlates with the serum-derived matrix stabilizing factor I{alpha}I and the hyaladherin family member TSG-6 (16). TSG-6, in turn, can bind chondroitin-4 sulfate, which constitutes the GAG side chains of versican (18). Of note, expression of TSG-6 is reduced in the COX-2 null mice (60) that show defective expansion of the COC matrix, thought to be the primary cause of ovulatory failure in these mice (6). Because we found that versican protein localized to the COC matrix of normal mice (Fig. 6Go), we also investigated its localization in COX-2 null ovaries. Versican localized to COC of ovulating follicles whether normally expanded in COX-2+/- or defective for expansion and ovulation in COX-2-/- mice. Thus, binding sites for versican in the expanded COC (HA?) are normally present in COX-2 null mice.

The role for versican in the theca interna is uncertain. Its presence in this region from 8–24 h after hCG treatment coincides with a time of neovascularization within this compartment and the formation of a vascular bed that is critical to structural as well as functional formation of CL (61). In contrast, versican is low/absent once luteinization and the vasculature that supports this tissue is established; i.e. in CL of d 7 pregnant mice (not shown). Versican V0 and V1 isoforms are involved in antiadhesive and promigratory effects during neovascularization (54). In addition, versican is present within the matrix that surrounds vascular smooth muscle and provides elasticity to that tissue (54, 62). Thus, versican localized to the CL periphery may be an important facilitator of vascular development or infiltration.

Proteolytic degradation in vitro of human versican by recombinant ADAMTS-1 through binding and cleavage of the ßGAG (E441–A442 of V1) domain was recently demonstrated (36). As mentioned above, we have shown that ADAMTS-1 is also acutely and PR dependently induced in ovulatory follicles (31, 49). It is interesting to speculate that ADAMTS-1 may cleave versican in one or both of the compartments of localization during ovulation. In support of this possibility, an approximately 70-kDa immunoreactive band was detected specifically in Western blot of isolated COC, and we have detected ADAMTS-1 immunolocalization within this same expanded matrix surrounding the COC (Russell, D. L., K. H. Doyle, S. Ochsner, J. D. Sandy, and J. S. Richards manuscript in preparation). The unaltered localization of versican in ADAMTS-1-deficient PRKO ovaries suggests that, if it is an endogenous substrate of ADAMTS-1 in the ovulating follicle, cleavage by this protease does not regulate localization of immunoreactive versican, but may generate products that may have specific or additional functions to the intact molecule (36).

These results demonstrate that versican V0, V1, and V3 mRNA and protein is a product of the developing ovarian follicle and up-regulated during ovulation. Expression and localization of versican is not dependent on crucial ovulatory genes PR or COX-2. However, the function of versican may be compromised in the PRKO mice due to the lack of ADAMTS-1. The localization of versican protein suggests that it may play roles in early stages of follicle organization as well as expansion of the cumulus matrix and vascularization or restructuring of the ovulated follicle wall. Clearly, much more needs to be learned about the interactions and functions of HA, versican, TSG-6, and ADAMTS-1 in the ovulating follicle as well as the specific role of versican in the small growing follicles where expression of HA, TSG-6, and ADAMTS-1 have not yet been detected.


   Acknowledgments
 
The authors thank Dr. Richard LeBaron (University of Texas San Antonio) for generously providing the Vc antibody used in Western blot and immunohistochemical analysis and Dr. John Sandy for helpful comments and discussion of the manuscript.


   Footnotes
 
This work was supported by NIH Grants HD-16629 and HD-07495 (to J.S.R.).

Abbreviations: ADAMTS, A disintegrin and metalloprotease with thrombospondin motifs; CL, corpora lutea/corpus luteum; COC, cumulus oocyte complex; COX-2, cyclooxygenase-2; Fo, forskolin; GAG, glycosaminoglycan; H, hypophysectomized; HA, hyaluronan; HAS-2, HA synthase-2; hCG, human chorionic gonadotropin; HE, H rats treated with 17-ß estradiol; HEF, HE rats treated with FSH; I{alpha}I, inter-{alpha}-trypsin inhibitor; NRS, normal rabbit serum; PMA, phorbol myristate acetate; PMSG, pregnant mare serum gonadotropin; PR, progesterone receptor; PRKO, PR knockout; RT, reverse transcriptase; SSC, sodium chloride/sodium citrate; T, testosterone; TSG-6, TNF{alpha}-stimulated gene 6; V0, V1, and V3, versican isoforms; Vc, rabbit anti-versican antibody.

Received April 23, 2002.

Accepted for publication November 8, 2002.


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 Discussion
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