Cloning and Characterization of a Rat Ortholog of MMP-23 (Matrix Metalloproteinase-23), a Unique Type of Membrane-Anchored Matrix Metalloproteinase and Conditioned Switching of Its Expression during the Ovarian Follicular Development

Junji Ohnishi, Eriko Ohnishi, Mulan Jin, Wakako Hirano, Dai Nakane, Hitoshi Matsui, Atsushi Kimura, Hirofumi Sawa, Kazuhisa Nakayama, Hiroshi Shibuya, Kazuo Nagashima and Takayuki Takahashi

Division of Biological Sciences (J.O., W.H., D.N., H.M., A.K., T.T.) Graduate School of Science Hokkaido University Sapporo 060-0810, Japan
Laboratory of Molecular and Cellular Pathology (E.O., M.J., H.S., K.N.) Hokkaido University School of Medicine CREST, JST (Japan Science and Technology) Sapporo 060-8638, Japan
Institute of Biological Sciences and Gene Experimental Center (K.N.) Tsukuba University Ibaraki 305-8572, Japan
Division of Morphogenesis (H.S.) Department of Developmental Biology National Institute for Basic Biology Okazaki 444-8585, Japan


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In our attempt to study the role of matrix metalloproteinases (MMPs) in the process of mammalian ovulation, we isolated a rat ortholog of the recently reported human MMP-23 from gonadotropin-primed immature rat ovaries. Transient expression of epitope-tagged rat and human MMP-23 in COS-1 cells revealed that they were synthesized as a membrane-anchored glycoprotein with type II topology. Indirect immunofluorescent analysis showed that subcellular localization of MMP-23 was predominantly in the perinuclear regions. The transfected human MMP-23 protein was processed endogenously to the soluble form in COS-1 cells. However, cotransfection of MMP-23 with the mouse furin cDNA did not enhance this processing, indicating that furin may not be involved in this event. Notably, in situ hybridization analysis revealed a dramatic switching of MMP-23 mRNA localization from granulosa cells to theca-externa/fibroblasts and ovarian surface epithelium during the follicular development. In serum-free primary culture of rat granulosa cells, a drastic diminution of MMP-23 mRNA expression was observed in response to FSH action between 24 h and 48 h of culture. The observed effect of FSH on MMP-23 expression was mimicked by treatment of granulosa cells with forskolin or 8-bromo (Br)-cAMP. In contrast, MMP-23 mRNA levels increased in theca-interstitial cells regardless of the presence of LH in the culture. However, treatment of theca-interstitial cells with forskolin or 8-Br-cAMP markedly reduced the expression of MMP-23 with a concomitant increase in progesterone production. These results indicate that the MMP-23 gene is spatially and temporally regulated in a cell type-specific manner in ovary via the cAMP signaling pathway.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Controlled regulation of extracellular matrix (ECM) composition represents an extremely important event in many normal and pathological processes. One major proteinase family, matrix metalloproteinases (MMPs), has been known to play a central role in degrading and remodeling components of ECM. Many MMPs are expressed widely during the development and morphogenesis of embryos to regulate ECM composition and influence basic cellular processes such as proliferation, migration, and differentiation (1). Under normal circumstances in the adult, MMPs are not produced in most cell types except neutrophils, and are not synthesized and secreted until biological demand occurs, e.g. wound healing, angiogenesis, arthritis, inflammatory diseases, and tumor invasion and metastasis (2, 3, 4).

Currently, the number of members of the MMP gene family has been growing, and at present more than 20 genetically distinct human MMPs have already been cloned and characterized. They can be classified into two major subfamilies according to their primary structure, and into six subfamilies according to their substrate specificity (5, 6). Based on their primary structural features, one is the soluble type of MMP, and the other is the transmembrane-type MMP (MT-MMP). Subfamilies based on the substrate specificity, consist of: (I) collagenases: collagenase-1, -2, and -3 (MMP-1, -8, and -13, respectively); (II) gelatinases: gelatinase A and B (MMP-2 and -9, respectively); (III) stromelysins: stromelysin-1 and -2 (MMP-3 and -10, respectively); (IV) stromelysin-like: matrilysin (MMP-7), metalloelastase (MMP-12) and MMP-19, (V) MT-MMPs: MT1, 2, 3, 4-MMP (MMP-14, -15, -16, and -17, respectively), MT5, 6-MMP (MMP-24, -25, respectively); (VI) others: stromelysin-3 (MMP-11) and enamelysin (MMP-20). All of the reported MMP members contain two typical common domain structures: the cysteine switch region of the propeptide and the zinc-binding site of the catalytic domain. Furthermore, another superfamily has been designated as the metzincin family, which contains the MMP family, members of the ADAM (a disintegrin and metalloprotease) family, and snake venom metalloproteinases with the distinctive zinc-binding consensus motif HExxH (7).

The adult female reproductive tract in mammals is a uniquely dynamic organism in which rapid and extensive degree of tissue development and tissue remodeling occur normally during each estrous cycle. MMPs are believed to be primary contributors to this matrix remodeling and are expressed in a highly regulated manner in the process of reproductive events, including menstruation, ovulation, implantation, and postpartum uterine involution (8). To gain insight into the role of MMPs in the tissue remodeling processes of the female reproductive organs, we performed RT-PCR with a pair of degenerate primers designed for the two conserved regions of the MMP family using RNA isolated from gonadotropin-primed immature rat ovaries. Six different MMP cDNAs were cloned including a MMP-like clone not yet identified during the course of the project. Due to its predominant expression in both rat and human female reproductive tract, we tentatively named it MIFR (metalloproteinase in the female reproductive tract) with the GenBank accession numbers of AB010960 and AB010961 for rat and human clones, respectively (9, 10, 11, 26).1 Recently, the human homolog of this clone designated as MMP-21/22 (9) and MMP-23 (10) and the mouse counterpart termed CA-MMP (11) were also reported. At present these clones have been designated as MMP-23 (6). In this report, we describe the molecular cloning and expression of the membrane-anchored type of rat MMP-23, which shows several unique structural features and an expression pattern distinct from all other MMPs so far characterized. Interestingly, in situ hybridization analysis revealed a conditioned switching of MMP-23 gene expression from granulosa cells to theca externa/fibroblasts in rat ovarian follicles, which depends on the state of follicle maturation in response to gonadotropin action. Furthermore, using serum-free cultures of rat granulosa cells and theca-interstitial cells, we showed that the MMP-23 gene expression was regulated in a cell type-specific manner during gonadotropin-induced differentiation via the cAMP signaling pathway.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Amplification of Fragments of the Rat Ovarian MMP cDNAs
To examine the expression of MMPs in rat ovaries during the periovulatory period, we initially used an immature animal model. Twenty-five-day-old rats were primed with equine CG (eCG) for 48 h to initiate follicle development and were then stimulated with human CG (hCG) to induce ovulation. This occurs 10–16 h after hCG administration. Total RNA was prepared from ovaries at 0, 2, 4, 8, and 12 h after hCG injection. RT-PCR was performed on each of these RNAs with a pair of degenerate primers designed for two highly conserved sequences of the MMP superfamily domains: the cysteine switch region and the zinc-binding domain. Amplified cDNA fragments of expected sizes were isolated from each reaction, pooled, and subcloned into a pBluescript vector. One hundred clones were randomly selected and sequenced. This allowed us to obtain cDNA fragments for six different MMP genes: collagenase-3 (MMP-13), gelatinase A (MMP-2), stromelysin-1 (MMP-3), matrilysin (MMP-7), metalloelastase (MMP-12), and an as yet unidentified protein with a high degree of similarity to MMPs as judged from the results of BLAST search.

Isolation of Rat and Human MMP-23 cDNAs
A preliminary Northern blot analysis of various rat tissues was conducted using a 498-bp fragment of the novel MMP-like gene as a probe. The result indicated that the corresponding mRNA transcript migrated as a single 1.4-kb transcript and was found to be expressed at the highest levels in ovary and uterus. We therefore decided to use the rat ovary for further cDNA cloning experiments. The isolated clone of 1,444-bp length appeared to be full-length and to potentially encode a 391-amino acid protein (Fig. 1AGo). The deduced amino acid sequence of the rat clone showed that it contained the sequence from Gly53 to Leu218 corresponding to the 498-bp RT-PCR amplified MMP-like product. This sequence included the consensus sequence HExGHxx involved in the zinc binding at the catalytic site of MMPs. However, another conserved sequence, the so-called cysteine switch, located in the profragment, was not found at positions 53 to 59 or at any other positions within the amino-terminal region of the putative catalytic domain. Alignment of nucleotide sequences of the 1.4-kb cDNA and the 498-bp RT-PCR product revealed that the highly homologous sequences for annealing the forward primer used for the RT-PCR were located at nucleotides 331 to 351 within the 1.4-kb cDNA (Fig. 1CGo). It is postulated that a single thymine residue insertion at nucleotide 340 would shift the reading frame to cause the lack of a cysteine in this region. To further confirm this unique structural feature, a 1,265-bp cDNA, highly homologous to the rat sequence, was isolated from a human uterus 5'-STRETCH cDNA library (CLONTECH Laboratories, Inc., Palo Alto, CA). It contained an entire open reading frame that encodes a protein of 390 amino acids.



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Figure 1. Structure of Rat and Human MMP-23

A, Alignment of the deduced amino acid sequences of the rat and human MMP-23. Individual amino acids are shown in single letter code, and identical amino acid residues are indicated by the shaded boxes. The GenBank accession numbers for the rat and human cDNA sequences are AB010960 and AB010961, respectively. N-glycosylation sites are indicated by asterisks (*), the cysteine residues in the carboxyl terminus of the catalytic domain are shown by circles ({bullet}), and the proline residues of the proline-rich region are indicated by triangles ({blacktriangledown}). B, Schematic representation of MMP-23 domain arrangements. The characteristic regions of MMP-23 are boxed. The type II signal anchor (SA), putative proprotein convertases recognition site (RRRR), catalytic domain, cysteine-rich region (Cys-rich), proline-rich region (Pro), and the carboxyl terminus are shown. The positions of each region are indicated as the number above the box for the human MMP-23 and below for the rat, respectively. C, Sequence comparison of the forward primer designed toward the cysteine switch with the highly homologous region of MMP-23 cDNA. The top amino acid sequence represents the cysteine switch conserved in MMPs family and the lower nucleotides are the degenerate sequences used as the forward primer in RT-PCR. The positions of identical nucleotides of corresponding rat MMP-23 cDNA with the forward primer sequence are indicated by vertical lines. Asterisks indicate identical nucleotides in the corresponding region of the rat MMP-23 cDNA with the mouse counterpart CA-MMP (18 ) or the human cDNA.

 
Figure 1AGo shows an alignment of the deduced amino acid sequences of the isolated rat and human MMP-like clones, sharing 83% sequence identity. Both clones possess the conserved typical zinc-binding site: HExGHxxGxxHS in the putative catalytic domain and a methionine residue (Met220 in human and Met221 in rat) that is proposed as the "Met-turn," which plays an essential role in the structure of the active site of proteins in the Metzincin family (7). Additionally, the sequence alignment with other MMPs using the Clustal W program of MacVector software (Oxford Molecular Ltd., Campbell, CA) predicted that the structural zinc-binding site of these clones comprised three histidines and one aspartic acid [His149, 170, 180, Asp151 in human and His150, 171, 181, Asp152 in rat, respectively (12, 13)]. The most notable difference between the rat and human MMP-like clones was observed in the amino-terminal 76 amino acids with only 48% identity. Sequence alignment analysis indicated that the rat and human clones are closely related to each other, and that they belong to the MMP family. However, they exhibit a series of domain structures with unique features (Fig. 1BGo). First, unlike other reported MMPs, the rat and human MMP-like clones lack the cysteine switch consensus sequences. Second, the hydropathy analysis of both protein sequences reveals the absence of a recognizable signal sequence at the amino-terminal end. Instead, the positively charged amino acid arginine (Arg19 in human and Arg18 in rat) was located at the amino terminus of the hydrophobic stretch at amino acids 20–39 and 19–38 in the human and rat sequences, respectively.

Since the mRNA is predominantly expressed in ovary and uterus (Fig. 2Go, B–D), we originally termed both rat and human cDNA sequences as MIFR and deposited these as such in the GenBank data base [accession numbers AB010960 and AB010961, respectively (9, 10, 11, 28)]. Although ovary and uterus are the two major sites of expression of this protein, significant levels of these mRNAs were also detected in the human male reproductive tract, including testis and prostate (Fig. 2DGo). During the course of this project, the human homolog of this clone named MMP-21/22 (9) and MMP-23 (10) and the mouse counterpart, designated CA-MMP (11), were reported. At present, all of these reported clones including MIFR have been designated as MMP-23 (6).



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Figure 2. Temporal Expression of the Rat MMP-23 mRNAs during the Periovulatory Periods and Chromosomal Localization of Human MMP-23

Temporal expression of MMP-23 and rpL32 in the gonadotropin-primed immature rat ovaries was analyzed by multiprobe RNase protection assays as described in Materials and Methods. Protected RNA fragments are indicated by arrowheads (A). Total RNA (20 µg per lane) from various rat tissues was electrophoresed and transferred to nylon membranes (NYTRAN, Schleicher & Schuell, Inc., Keene, NH). The blotted membranes were hybridized with a 32P-labeled full-length rat MMP-23 cDNA and exposed for 7 days or 1 month to x-ray films (X-OMAT, Eastman Kodak Co., Rochester, NY) with intensifier screens (B). Commercially obtained filters (Human Multiple Tissue Blot, CLONTECH Laboratories, Inc.) containing 2 µg poly (A)+ RNA per lane were probed with a 32P-labeled full-length human cDNA. The filters were exposed to x-ray films (Bio-Max, Eastman Kodak Co.) with intensifier screen for 24 h (C) or 4 days (D). The position of the human MMP-23 gene was determined by PCR analysis of radiation hybrid panel DNAs (Stanford G3 panel). The described chromosome 1 ideogram is courtesy of Drs. Rick Myers and David Cox at Stanford Genomic Center (E).

 
Kinetics of the MMP-23 mRNA Expression during the Periovulatory Period
To determine the kinetics of MMP-23 expression during the periovulatory period, a detailed time course experiment was performed by multiprobe RNase protection assay using 20 µg of total RNA obtained from ovaries of eCG- and hCG-treated rats. Figure 2AGo shows that the MMP-23 was constitutively expressed throughout the periovulatory period even in the immature state.

Function of the Hydrophobic Stretch Present in the Amino Terminus of MMP-23
Using the program of ExPASY via Internet at http://www.expasy.ch/tools/#transmem, it is predicted that the hydrophobic stretch of MMP-23 may serve as a type II signal anchor (14, 15, 16). To analyze this possibility, we constructed cytomegalovirus (CMV)-driven expression plasmids with the epitope-tagged sequences at the carboxyl-terminal end of MMP-23 (Fig. 3AGo). As analyzed by Western blotting experiments using the monoclonal antibodies 3F10 for hemagglutinin (HA) epitope tag and M2 for FLAG tag, both human and rat MMP-23 were detected in membrane fractions prepared from transiently transfected COS-1 cells (Fig. 3BGo, lanes 4 and 5 and 12 and 13). Epitope-tagged proteins were synthesized as 51 kDa and 56 kDa forms in rat and human, respectively, both being larger than those theoretically calculated from the primary structure (46 kDa and 45 kDa for rat and human, respectively). Reduction of the molecular size of both proteins by endoglycosidase H treatment indicates that differences in molecular size reflect the glycosylation of MMP-23 (Fig. 3BGo, lanes 7 and 8 and 15 and 16). These data clearly showed, for the first time, that rat and human MMP-23 were both synthesized as membrane-associated glycoproteins with type II topology.



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Figure 3. Function of the Potential Type II Signal Anchor at the Amino Terminus of MMP-23

A, Schematic structures of the rat and human MMP-23 constructs tagged with the HA and FLAG epitope, respectively. Dark colored boxes represent hydrophobic stretches. Putative proprotein convertases recognition site and the catalytic domain are shown as RRRR and HEIGHALGLMHSQ, respectively. B, Transient expression of the epitope-tagged MMP-23 in COS-1 cells. Concentrated media and the membrane fractions were prepared from COS-1 cells transiently transfected with various expression plasmids: the HA-tagged rat MMP-23 with a control plasmid (pcDNA3, lanes 1 and 4) or mouse furin expression plasmid (lanes 2 and 5), a control plasmid with mouse furin (lanes 3 and 6 and 11 and 14), and the FLAG-tagged human MMP-23 with a control (lanes 9 and 12) or mouse furin (lanes 10 and 13). Lanes 1–3 and 9–11 are concentrated conditioned media, and lanes 4–8 and 12–16 are membrane fractions. The solubilized membrane fractions were treated overnight with (lanes 8 and 16) or without (lanes 7 and 15) endoglycosidase H (Endo.H). The corresponding amounts of the samples were subjected to 10% SDS-PAGE under reducing conditions and immunoblotted with anti-HA antibody 3F10 or anti-FLAG antibody M2. Arrowheads indicate glycosylated and deglycosylated forms of rat and human MMP-23, and molecular masses are described in kilodaltons. C, Immunofluorescence localization of epitope-tagged MMP-23 transiently expressed in COS-1 cells. Both HA-tagged rat MMP-23 and FLAG-tagged human MMP-23 were detected specifically in the perinuclear regions and in the periphery of the cells with 3F10 and M2 antibody, respectively.

 
To evaluate the subcellular localization of epitope-tagged MMP-23 expressed in COS-1 cells, indirect immunofluorescent staining analysis was performed with anti-HA or FLAG antibodies on fixed and permeabilized cells. Figure 3CGo shows strong staining signals for both HA and FLAG epitopes in the perinuclear regions that could correspond to endoplasmic reticulum, the Golgi, or endosomal/lysosomal compartments. In addition, distinctive punctuate staining was observed in the periphery of the cells, on or near the cell surface. No significant signals were detected in mock transfected cells used as negative controls (data not shown).

Cleavage Analysis of MMP-23 by Furin
Both rat and human MMP-23 possess the unique dibasic motif (RRRR), a putative recognition site for furin (17), between the type II signal anchor and the catalytic domain. This sequence was previously identified and proven to mediate the intracellular activation of pro-MT-MMPs (18, 19), prostromelysin-3 (20, 21, 22), and ADAMs (23) in a furin-dependent manner. Accordingly, it could be anticipated that MMP-23 is converted intracellularly from a membrane-anchored form to its soluble form by a proprotein convertase or by ectodomain shedding. Indeed, a soluble form of the FLAG-tagged human MMP-23 (54 kDa) was processed into the culture medium by COS-1 cells (Fig. 3BGo, lanes 9 and 10). In contrast, no significant soluble form of the HA-tagged rat MMP-23 was detected (Fig. 3BGo, lanes 1 and 2). Moreover, coexpression of mouse furin with the epitope-tagged MMP-23 did not enhance the formation of the soluble form, indicating that furin may not be involved in this event (Fig. 3BGo, lanes 2 and 10). Nevertheless, no secreted form of human MMP-23 was detected in the culture medium of the mutant in which the predicted furin cleavage site was eliminated by changing the critical amino acids RRRR78 to RRNG78 by site-directed mutagenesis (data not shown). This result indicated that the RRRR78 motif was required for human MMP-23 secretion.

Chromosomal Mapping of Human MMP-23
By using the Stanford G3 radiation hybrid panel, the human MMP-23 gene was determined to be located at D1S2565 (SHGC-4723) within the terminal end of the short arm of chromosome 1, placing it between the markers D1S243 and D1S253 within the band 1p36.3 (Fig. 2EGo; Ref. 24). The obtained result is well in agreement with two recent reports by Gururajan et al. (9) and Velasco et al. (10).

Differential Distribution of MMP-23 in the Rat Female Reproductive Tract
In situ hybridization analysis was performed to determine which cell types are responsible for the synthesis of MMP-23 in individual ovarian follicles, oviduct, and uterus during the periovulatory period. In ovaries of untreated immature rats (Fig. 4Go, A and B), MMP-23 expression was found to be strictly confined to granulosa cells (black arrow) of preantral and small antral follicles. No significant signal was observed in theca-interstitial cells during these stages. When immature rats were administered eCG followed by hCG treatment, the follicles increased in size due to the proliferation of granulosa and theca cells and an enlargement of the antrum (Fig. 4Go, C–F). During the transition from the small antral to the large antral follicles, and developing corpus luteum, the intensity of the signal for MMP-23 mRNA in granulosa cells greatly diminished to a baseline level. Instead, strong staining for MMP-23 mRNA was observed in theca-externa/fibroblasts (black arrowhead, Fig. 4, D and F) and in ovarian surface epithelium (white arrowhead, Fig. 4F). Weak but clearly evident signal was also detected in part of the stroma of the ovary from hCG-treated rats.



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Figure 4. In Situ Localization of MMP-23 mRNA during the Various Stages of Follicular Development

Ovaries (A–F), uteri (G), and oviducts (H) from untreated immature rats or eCG- and hCG-treated rats were removed. Frozen sections were hybridized with DIG-labeled rat MMP-23 antisense RNA probe. The hybridization signal appears in dark purple. Sections A and B were from ovaries of untreated immature rats, and sections C–F were obtained at 6 h after hCG administration. Sections G and H were from uterus and oviduct of untreated immature rats, respectively. Black arrows indicate granulosa cells. Black and white arrowheads indicate theca-externa and ovarian surface epithelium, respectively (C–F). White arrows indicate stroma area of uterus (G) and oviduct (H). A, C, E, x8; B, D, F, x20; G, x40; H, x20.

 
MMP-23 localization in uterus and oviduct was also investigated in the present study. The stroma of all zones of oviduct (Fig. 4G) and endometrium (Fig. 4H) displayed high levels of MMP-23 mRNA expression with a fixed localization and a constant content of mRNAs throughout the gonadotropin-induced periovulatory periods (data not shown).

Hormonal Regulation of MMP-23 Gene Expression in Primary Cultures of Rat Granulosa Cells and Theca-Interstitial Cells
Serum-free primary cell cultures of rat granulosa cells and theca-interstitial cells were used to investigate the conditioned switching mechanism of MMP-23 expression observed in vivo. Both types of prepared cells showed a cell type-specific regulation in response to gonadotropins accompanied by the accumulation of steroid hormones and cAMP during differentiation. Treatment with FSH stimulated cAMP and progesterone production in granulosa cells (Fig. 5Go, A and B), while LH-induced differentiation of theca-interstitial cells caused a marked enhancement of cAMP and androstenedione synthesis. An LH dose of 5 ng/ml produced a response in thecal progesterone production. It should be mentioned that LH-treated theca-interstitial cells produced progesterone at levels lower than that observed for androstenedione at time points greater than 24 h (Fig. 5Go, E and F). Using these conditioned cells, we performed detailed time course studies of MMP-23 gene expression by semiquantitative multiplex RT-PCR. Figure 5Go, C and D, shows an accumulation of MMP-23 mRNA in untreated granulosa cells during 48 h of culture. FSH treatment of granulosa cells repressed MMP-23 expression after 24 h of culture and caused a drastic fall in mRNA accumulation up to approximately 80% below the control levels at 48 h. In contrast, MMP-23 mRNA levels increased in theca-interstitial cells regardless of the presence of LH during culture. However, MMP-23 mRNA levels in LH-treated cells were somewhat lower (~20%) than those of the unstimulated controls at any of the investigated periods (Fig. 5Go, G and H).



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Figure 5. Time Course of the Effect of Gonadotropins on MMP-23 mRNA Expression, cAMP Production, and Steroidogenesis in Serum-Free Cultured Rat Granulosa Cells and Theca-Interstitial Cells

Granulosa cells (3 x 105 viable cells per well, A–D) and purified theca-interstitial cells (2 x 105 viable cells per well, E–H) were cultured for 48 h in the presence of ITS-x supplement with (black symbol) or without (white symbol) FSH (50 ng/ml) and LH (5 ng/ml), respectively. Conditioned media collected at selected times from each treatment group were assayed for cAMP production (A and E), and the released steroid hormones (B and F). Data (means ± SEM) are from two independent experiments with quadruplicate incubations per experiment. Semiquantitative multiplex RT-PCR was performed for 30 cycle amplification using the total RNA prepared from each treatment group with MMP-23 primers and Quantum RNA alternate 18S internal standards. Representative PCR products stained with SYBR Green I were demonstrated in panels D and H. MMP-23 expression was normalized to expression of 18S rRNA presented as the ratio of fluorescence intensity in the MMP-23 and 18S bands (C and G). Data are shown as the mean ± SEM of three separate experiments with duplicate measurements.

 
cAMP-Generating System Is the Key Regulator of MMP-23 Gene Repression
It is well known that the action of both FSH and LH is mediated by cAMP via activation of adenylyl cyclase; therefore, we examined whether the repression of MMP-23 in response to gonadotropins is a cAMP-mediated phenomenon in rat ovarian cells. Mimicking the action of FSH on the differentiated granulosa cells, transcriptional levels of MMP-23 were remarkably attenuated by treatment with forskolin at 0.1 mM, which is a direct activator of adenylyl cyclase, or after treatment with the permeable cAMP analog 8-Br-cAMP at 1 mM for 48 h (Fig. 6Go, B and C). This was accompanied by the accumulation of progesterone in the conditioned medium (Fig. 6AGo). Notably, cultured theca- interstitial cells in the presence of forskolin at 0.01 mM or 8-Br-cAMP at 1 mM showed a drastic suppression (~80%) of MMP-23 transcription compared with that of the control (Fig. 6Go, E and F). Under the above conditions, the phenotype of theca-interstitial cells was switched from an androstenedione-producing state to the luteinizing condition with increased progesterone production (Fig. 6DGo).



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Figure 6. Effect of Forskolin and 8-Br-cAMP Treatment on MMP-23 mRNA Expression in Serum-Free Cultures of Rat Granulosa Cells and Theca-Interstitial Cells

Granulosa cells (3 x 105 viable cells per well, A–C) and purified theca-interstitial cells (2 x 105 viable cells per well, D–F) were cultured for 48 h in the presence of ITS-x supplement with or without stimulants: forskolin, 8-Br-cAMP, or 8-Br-cGMP. Conditioned media of granulosa cells were analyzed for progesterone production (A), and androstenedione (white bar) and progesterone (black bar) assay were performed with the media of theca-interstitial cells (D). Data are shown as the mean ± SEM of four separate experiments. Semiquantitative multiplex RT-PCR was performed for 30 cycles amplification using the total RNA prepared from each treatment group with MMP-23 primers and alternate 18S internal standards. Representative SYBR Green I-stained PCR products are illustrated demonstrated in panels C and F. MMP-23 expression was normalized to expression of 18S rRNA presented as the ratio of fluorescence intensity in the MMP-23 and 18S bands (B and E). Effects of each treatment on steroidogenesis and MMP-23 expression are represented by a vertical line in panels A–C and D–F.

 
Figure 7Go shows in situ hybridization analysis of the cultured cells confirming the observed effect of repression on MMP-23 expression with cytological views. MMP-23 expression was detected in all of the small round cells cultured in the absence of FSH (Fig. 7AGo). A marked repression of MMP-23 was confirmed by FSH treatment accompanied by morphological changes to the fully rounded appearance (Fig. 7CGo). In the case of theca-interstitial cells, a subset of the prepared cells, those with a slightly round or flattened shape, were responsible for the MMP-23 expression (Fig. 7EGo). No morphological changes in theca-interstitial cells were observed during the 5 ng/ml LH-induced differentiation (data not shown), while forskolin treatment caused the morphological changes of stretched shape, and completely repressed MMP-23 expression down to background levels (Fig. 7GGo).



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Figure 7. Effects of FSH or Forskolin on MMP-23 mRNA Levels in Cultured Granulosa Cells and Theca-Interstitial Cells

Granulosa cells (2 x 105 cells) and theca-interstitial cells (1 x 105 cells) were prepared as described in Materials and Methods. Effects of FSH (50 ng/ml) and forskolin (0.01 mM) on MMP-23 mRNA expression in granulosa cells (C and D) and in theca-interstitial cells (G and H), respectively, were examined by in situ hybridization. Effects of these treatments on each type of the cells were compared with the control cells cultured in the absence of FSH (A and B) or forskolin (E and F). Upper panels (A, C, E, and G) were hybridized with the antisense probe of rat MMP-23 and lower panels (B, D, F, and H) were hybridized with the sense probe. A–D, x200; E–H, x100.

 
Immunohistochemical Studies of MMP-23 in the Rat Ovary
To address the localization of MMP-23 protein in the ovary, we first developed an anti-MMP-23 antibody. A polyclonal antiserum was raised against the synthetic peptide corresponding to amino acids 329–342 of human MMP-23. The same sequence is located at amino acids 331–344 in the rat ortholog. As expected, the obtained GN2062 antiserum recognized both epitope-tagged human and rat MMP-23 transiently expressed in COS-1 cells (Fig. 8Go).



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Figure 8. Immunohistochemical Analysis of MMP-23 Localization in eCG/hCG-Primed Rat Ovary

Antihuman MMP-23 polyclonal antibodies were raised against a synthetic peptide with 14 residues in the carboxyl-terminal domain of human MMP-23. A 1:1,000 dilution of the obtained antiserum GN2062 was demonstrated by Western blotting to recognize specifically both human and rat epitope-tagged MMP-23 expressed in the membrane fractions of COS-1 cells.

 
Using this antiserum, immunohistochemical analysis was performed to detect the localization of MMP-23 protein during the follicle maturation in the rat ovary. In 25-day- old immature rat ovaries, MMP-23 protein in preantral and small antral follicles was widely distributed in granulosa cells, corresponding to regions of active mRNA synthesis. Notably, intense signals for MMP-23 protein were also observed in the theca-interna region of these follicles, in which no transcript of MMP-23 was detected (Fig. 9Go, A and C). During follicle maturation after hCG administration, the intensity of immunopositive signals of MMP-23 was relatively weaker than that of immature ovary, although a similar pattern of protein localization was observed. Unexpectedly, distinct localization patterns were observed for MMP-23 protein and mRNA in large antral follicles. Theca-externa/fibroblasts exhibiting robust MMP-23 mRNA expression in antral follicles showed very weak immunoreactive signals. Stronger signals were observed in theca-interna. Expression of MMP-23 mRNA was repressed in granulosa cells, while its protein was still detectable (Fig. 10Go, A and C). The specificity of immunostaining was confirmed by the complete abolition of signals by using preimmune serum (Figs. 9BGo and 10BGo) or after preincubation of the antiserum with a saturating concentration of synthetic MMP-23 peptide.



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Figure 9. Immunohistochemistry and in Situ Hybridization of MMP-23 in Serial Sections of Immature 25-Day-Old Rat Ovary

Immunohistochemical studies were performed on 10 µm periodate-lysine-paraformaldehyde-fixed frozen sections of ovary (A and B). Specificity of staining was determined by comparison with the serial sections incubated with an equal amount of nonimmunized rabbit serum (B) or with a specific antiserum, GN2062, neutralized by preabsorbing an antiserum with a 20-fold (by weight) excess of the antigen peptide. In 25-day-old rat ovaries, immunoreactive signals for MMP-23 were observed in both granulosa cells (black arrowhead) and theca-interna (white arrowhead) of preantral and small antral follicles (A). In situ localization of MMP-23 mRNA was shown to be restricted to granulosa cells (C). Panel D shows the hematoxylin and eosin staining of the serial section.

 


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Figure 10. Immunohistochemistry and in Situ Hybridization of MMP-23 in Serial Sections of eCG Plus hCG-Primed Rat Ovary

Twenty four-day-old immature rats were primed with 10 IU eCG and administered with 10 IU hCG 48 h after eCG injection. Ovaries were collected 6 h after hCG treatment. Significant immunopositive signals for MMP-23 were observed in granulosa cells (black arrowhead) and theca-interna (white arrowhead) of large antral follicles and some parts of newly formed corpus luteum (asterisk) in the ovary (A). Much less immunopositive signals were detected in theca-externa/fibroblasts (white arrow), which were the major sites of MMP-23 mRNA production (black arrow, panel C). Panel D shows the hematoxylin and eosin staining of the serial section.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Matrix metalloproteinases (MMPs) have been recognized as crucial role players in establishing and maintaining a proper ECM environment in the female reproductive tract via their distinct substrate specificity and their spatial and temporal expression profiles. We report here the cDNA cloning of the rat ortholog MMP-23, which shows a unique type of membrane- anchored MMP. Additionally, we demonstrated that MMP-23 expression is regulated in a cell-type specific manner during ovarian follicle maturation. From its unique structural features, MMP-23 can be classified into another group of membrane-type MMP, which anchors with type II topology in plasma membranes via a hydrophobic domain near the amino terminus. As shown in Fig. 1CGo, the lack of a cysteine switch in MMP-23 can be explained by a frame shift due to a single thymine insertion into this conserved sequence in the process of evolution. These observations lead us to speculate that the MMP-23 gene may have recently branched off from the MMP tree by sequence alteration occurring in exon 1 that encodes this region.

Transient expression experiments of an epitope-tagged MMP-23 revealed that the unique dibasic motif might not function as a recognition site for furin. This is comparable to a previous example reported by Cao et al. (25) in which coexpression of the recombinant full-length membrane-bound MT1-MMP with furin in COS-1 cells had no effect on the processing of the membrane-associated proteinase. However, we found that the soluble form of FLAG-tagged human MMP-23 was processed endogenously in COS-1 cells and that processing was definitely abolished by introducing the mutation into the RRRR78 motif, suggesting this proprotein convertase recognition site is required for MMP-23 secretion. Recently, Pei et al. (26) provided direct evidence that the mouse counterpart CA-MMP/MMP-23 is processed at the RRRR79 motif before being secreted by MDCK cells stably expressing the FLAG-tagged recombinant protein. Alternatively, another proprotein convertase, e.g. PACE4, PC4, PC6A, PC6B, or a potential candidate protease, may be required for the efficient processing of MMP-23 (17).

Glycosylated forms of both rat and human MMP-23 were demonstrated to be highly sensitive to endoglycosidase H and representative of the high mannose-type glycoproteins. Consequently, overexpression of MMP-23 in COS-1 cells may cause an impaired processing of the mannose-rich N-linked glycan proteins in the Golgi, leading to the inefficient conversion of the membrane-anchored forms into the soluble species by proprotein convertases in the trans-Golgi network, which usually accompanies the secretion of MMP-23. This may be one possible explanation for the differences in the amount of the recombinant MMP-23 secretion that was observed for the rat and human proteins. Failure to detect the soluble form of rat recombinant protein may have resulted from a large intracellular accumulation of immature endoglycosidase H-sensitive protein, since much higher levels of the rat protein expression were observed than that of the human in COS-1 cells (Fig. 8Go). Otherwise, there may exist some cell type-specific factors or mechanisms that facilitate the intracellular targeting and processing of MMP-23 (27). Our current efforts to clarify detailed molecular events of processing and secretion of MMP-23 have been performed using several ovarian cell lines endogenously expressing MMP-23.

Immunohistochemical analysis of rat MMP-23 in the ovary demonstrated the distinct localization patterns of MMP-23 protein and its mRNA during the follicle maturation (Figs. 9Go and 10Go). Notably, significant levels of immunopositive signals for MMP-23 were observed in theca-interna, which did not correspond to regions of mRNA synthesis. On the other hand, theca-externa/fibroblasts expressing high levels of MMP-23 mRNA in antral follicles showed very weak immunoreactive signals. These results may provide indirect evidence that membrane-anchored MMP-23 is processed in vivo to the soluble form and is secreted and diffused through extracellular spaces in the follicles.

In situ hybridization analysis revealed a conditioned switching of MMP-23 gene expression in the ovary of gonadotropin-primed immature rats (Fig. 4Go, A–F), whereas the RNase protection assay demonstrated that expression levels of MMP-23 transcripts were apparently constant at various stages of follicular development (Fig. 2AGo). These results clearly indicate that the expression of MMP-23 mRNA is likely to be regulated spatially and temporally by gonadotropins in the ovary and that this regulation is associated with the state of the development of each follicle. This in vivo phenomenon was confirmed by in vitro experiments using primary cultures of granulosa cells and theca-interstitial cells prepared from immature ovaries. The down-regulation of MMP-23 expression in granulosa cells induced by FSH (Fig. 5Go, C and D) and the accumulation of its mRNA in LH-treated theca-interstitial cells in vitro (Fig. 5Go, G and H) was found to be in good agreement with the in vivo results demonstrated by in situ hybridization (Fig. 4Go, A–F). These results indicate that the serum-free culture system of ovarian cells used in this study is the preferable in vitro tool to reflect and characterize in vivo profiles of MMP-23 expression.

A note of caution must be made concerning the accumulation of MMP-23 mRNA observed in theca-interstitial cells regardless of the presence of LH during culture, despite the fact that LH treatment showed slightly repressive effect on MMP-23 expression. The level of MMP-23 repression by LH treatment was evidently much lower than that observed in forskolin or 8-Br-cAMP treated cells (Fig. 5Go, G and H, and Fig. 6Go, E and F). Additionally, Fig. 6Go, E and F, showed that 8-Br-cAMP repressed MMP-23 expression in theca-interstitial cells, in a dose-dependent manner. These results may have been caused by the quite low level of LH receptor expressed in theca-externa/fibroblasts corresponding to the site of MMP-23 synthesis (28, 29). Unfortunately this is difficult to prove because of the technical difficulty in separating theca-interna and theca-externa at present. Although, it can be postulated that LH could not produce sufficient amounts of cAMP in theca-externa/fibroblasts to repress MMP-23 expression to a level similar to that observed in the forskolin or 8-Br-cAMP-treated cells. In both types of the granulosa cells and theca-interstitial cells, 8-Br-cGMP essentially had no effect on MMP-23 gene expression (Fig. 6Go, B, C, E, and F), indicating that at least the transcriptional repression of MMP-23 is directly associated with the cAMP signaling pathways.

Previous reports have demonstrated that progesterone represses MMP-1, -3, -7, -9 and -11 mRNA in cultured endometrial tissue (8). As shown in Fig. 5Go, B–D, the time course of MMP-23 repression apparently coincided with that of progesterone synthesis in FSH-treated granulosa cells. As evidenced by the observation in theca-interstitial cells (Fig. 6Go, D–F), a degree to which MMP-23 expression was repressed seems to be correlated with the increased extracellular progesterone levels. However, in the present study no significant effects on MMP-23 expression were observed in either granulosa or theca-interstitial cells cultured for 48 h in the presence of progesterone or the progesterone antagonist mifepristone (RU486, data not shown). Furthermore, it is well recognized that progesterone receptor expression is selectively induced and localized in granulosa cells, not theca-interstitial cells of preovulatory follicles within 5–7 h of the LH surge in rats (30, 31). A more indirect role for progesterone may not be excluded, although it is unlikely that the observed transcriptional repression of MMP-23 in both granulosa cells and theca-interstitial cells could be attributable to the direct action of progesterone on the MMP-23 gene regulatory elements through the progesterone nuclear receptor.

It is noteworthy that a marked elevation of MMP-23 transcripts was observed in untreated granulosa cells and theca-interstitial cells cultured for 48 h, indicating that an autonomous system to enhance MMP-23 gene expression is present in both types of cells. Interestingly, similar expression profiles have been reported as an example of angiotensin II type 2 receptor expression in the granulosa cells cultured in the absence of FSH (32, 33). Since culturing the granulosa cells in serum-free conditions without FSH has been shown to lead to the onset of spontaneous apoptosis, it may be proposed that MMP-23 plays some role in the formation and/or maintenance of atretic follicles (33).

The exact nature of the conditioned switching mechanism of MMP-23 gene expression during follicle maturation remains unclear, but this complex expression pattern could indicate that MMP-23 has different roles in granulosa cells and theca cells of the follicle during its development, ovulation, and corpus luteum formation. Additionally, the presence of high levels of MMP-23 transcripts in primary follicles as early as 7 days after birth indicates that this proteinase in granulosa cells may be involved in early follicular development (E. Ohnishi and J. Ohnishi, unpublished data). The molecular mechanisms underlying the ovarian cell type-specific profile of MMP-23 gene expression is currently under intense investigation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hormones
Ovine FSH (NIDDK-oFSH-18:6xNIH-oFSH-S1, 1640IU/mg, 0.1xNIH-oLH-S1, 106IU/mg) and ovine LH (NIDDK-oLH-26 or AFP-5551B: 2.3IU/mg) were obtained through the National Hormone and Pituitary Program, NIDDK, and Dr. A. F. Parlow (Torrance, CA). Diethylstilbestrol, sesame oil, and Mifepristone (RU486) were purchased from Sigma (St. Louis, MO). Modified McCoy’s 5A medium, Hank’s balanced MEM, and HBSS were purchased from Life Technologies, Inc. (Gaithersburg, MD). 8-Br-cAMP, 8-Br-cGMP, and forskolin were obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan).

Animals
Immature (21-day-old) female Sprague Dawley rats were purchased from Charles River Breeding Laboratory (Yokohama, Japan) and housed at a constant temperature on a 14-h light, 10-h dark cycle and fed rat chow and tap water ad libitum. At 25 days of age, the rats were injected sc with 10 IU of eCG (Sigma) to initiate follicle development and 48 h later with 10 IU of hCG (Sigma) to induce ovulation. Ovulation took place 10–16 h after hCG administration. While under deep halothane/fluorothane (Takeda Chemical Industries, Ltd., Osaka, Japan) anesthesia, animals were killed by decapitation at different intervals after hormone treatment, and ovaries and uteri were quickly removed for RNA preparation and in situ hybridization. Animals were treated in accordance with the principles and procedures outlined in the "Guidelines for Care and Use of Experimental Animals," as approved by the Animal Care and Use Committee at Hokkaido University School of Medicine.

RT-PCR
cDNAs were synthesized using 5 µg of total RNA, which was prepared from ovaries removed at 0, 2, 4, 8, and 12 h after hCG injection, by SuperScript Preamplification System (Life Technologies, Inc.) and used as templates for the PCR performed with two degenerate oligonucleotides. The forward primer was 5'-(A/C)G(A/G/C)TGTGG(A/T)GT(C/G/T)CC(A/T/C)GATGT-3', and the reverse primer was 5'-AGGG(A/C)(G/A)TGGCCAA(G/A)(C/T)TCATG-3'. Both were designed for highly conserved sequences among the MMP family corresponding to the cysteine switch domain PRCG(N/V)PD and the zinc-binding region HExGHxx, respectively. The PCR was carried out with 40 cycles of denaturing (94 C, 1 min), annealing (55 C, 2 min), and extension (72 C, 3 min).

Multiprobe RNase Protection Assay
Multiprobe RNase protection assay was performed using RNase Protection Kit (Roche Molecular Biochemicals, Indianapolis, IN). To prepare cRNA probes, cDNA fragments of rat MMP-23 (nucleotides 532–1,049) and ribosomal protein L32 (nucleotides 237–380, X06483) were subcloned into appropriate sites of a pBluescript II SK (+) vector (Stratagene, La Jolla, CA), and transcribed in vitro in the antisense direction by T7 or T3 RNA polymerase in the presence of [{alpha}-32P]UTP using RNA Transcription Kit (Promega Corp., Madison, WI). Twenty micrograms of total RNA were incubated with two labeled cRNA probes (20,000~50,000 cpm per reaction for each probe) in a single tube at 60 C for 16 h.

cDNA Cloning and Sequence Analysis of MMP-23
Approximately 4.5 x 105 clones of rat ovarian cDNA libraries constructed in {lambda}gt10-EcoRI arms were screened by hybridization with a 32P-labeled 498-bp RT-PCR fragment of the novel MMP-related clone mentioned above. For isolating the human counterpart, approximately 7.0 x 105 plaques from the human uterus 5'-STRETCH cDNA library (CLONTECH Laboratories, Inc.) were screened with a 32P-labeled rat MMP-23 full-length cDNA.

Transient Expression of the Epitope-Tagged Rat and Human MMP-23
Carboxyl-terminally tagged expression plasmids of rat MMP-23 with an influenza virus hemagglutinin (HA) epitope sequence and human MMP-23 with a FLAG tag were constructed by ligating each cDNA fragment into pMH vector (Roche Molecular Biochemicals) and pCMVTag4 vector (Stratagene), respectively. The HA-tagged rat MMP-23 and FLAG-tagged human MMP-23 mutants at the putative furin-cleavage site were made by using the QuikChange site-directed mutagenesis kit (Stratagene). Constructed plasmids were prepared for transfections into COS-1 cells (Riken Cell Bank, Tsukuba, Japan) by using Endofree Plasmid Maxi Kit (QIAGEN, Chatsworth, CA). Transient transfections of the expression plasmids into COS-1 cells were performed using FuGENE6 (Roche Molecular Biochemicals) according to the manufacturer’s instructions. pcDNA3 (Invitrogen, San Diego, CA) was used to equalize the total amount of CMV-derived plasmids per each dish.

Indirect Immunofluorescence Analysis
COS-1 cells grown on glass coverslips were transfected with MMP-23 expression plasmids or pcDNA3 (MOCK) using FuGENE6 and cultured for 24 h in DMEM with 10% FBS. Cells were fixed with 3% paraformaldehyde in PBS for 30 min on ice, permeabilized with 0.1% Triton X-100 for 7 min on ice, and treated with 1 µg/ml rat anti-HA high affinity antibody 3F10 (Roche Molecular Biochemicals) or 20 µg/ml of mouse anti-FLAG M2 antibody (Stratagene) in HBSS containing 5% crystallized BSA (Wako Pure Chemical Industries Ltd.) for 1 h at room temperature. After washing with PBS, cells were incubated with Cy3-conjugated antirat or antimouse IgG (Amersham Pharmacia Biotech, Arlington Heights, IL) for 1 h at room temperature in HBBS containing 5% crystallized BSA. The coverslips were mounted with Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA) and observed on a fluorescence microscope.

Preparations of Membrane Fractions and Conditioned Medium, and Western Blotting
COS-1 cells were transiently transfected with either human or rat MMP-23 expression plasmid with or without mouse furin expression vector pCMVmFur (36) using FuGENE6. After 24 h culture with 10% FBS, cells were washed twice with serum-free DMEM/F12 and further cultured for 48 h with serum-free DMEM/F12 containing 15 mM HEPES, 1 x IST-X (10 mg/liter of insulin, 5.5 mg/liter of transferrin, 6.7 µg/liter of sodium selenite, and 2 mg/liter of ethanolamine, Life Technologies, Inc.) and 1 x lactoalbumin hydrolysate (Life Technologies, Inc.). The conditioned media were concentrated approximately 100-fold using Centricon-10 (Millipore Corp., Bedford, MA). To isolate membrane fractions, cells were lysed with 20 mM HEPES, pH 7.4, 0.25 M sucrose, 10 mM EDTA, 1 x Complete (Roche Molecular Biochemicals) for 30 min on ice, and then homogenized by a Dounce homogenizer. After centrifugation at 700 x g for 15 min at 4 C, the supernatant was centrifuged at 100,000 x g for 30 min at 4 C. The pellet was solubilized for 1 h on ice in RIPA buffer (Roche Molecular Biochemicals) and used for Western blotting analysis as the membrane fractions. Corresponding amounts of concentrated media and membrane fractions were electrophoresed in 10% SDS-polyacrylamide gels under reducing conditions and transferred to polyvinylidene difluoride membranes (Immobilon P, Millipore Corp.). Blotted proteins were probed with 3F10 antibody at 0.1 µg/ml or M2 antibody at 1 µg/ml, and specific bands were visualized using peroxidase-conjugated antirat or mouse IgG (Amersham Pharmacia Biotech) at 1:10,000. The ECL or ECL Plus Western Blotting Kit (Amersham Pharmacia Biotech) was used according to manufacturer’s directions.

For deglycosylation of epitope-tagged MMP-23, solubilized membrane fractions were treated with or without endoglycosidase H (Endo H, Roche Molecular Biochemicals) for 18 h at 37 C in 50 mM sodium acetate buffer (pH 5.5) containing 1% SDS and 0.1 M dithiothreitol according to the manufacturer’s protocol.

Cloning of the Human MMP-23 Gene and Genetic Mapping
Human genomic MMP-23 clones (GenBank accession number, AB031068) were obtained by screening the human cosmid library (CLONTECH Laboratories, Inc.) with the full length of human MMP-23 cDNA as a probe. To determine the MMP-23 chromosomal locus, the Stanford G3 RH panel with 83 radiation hybrid clones (Research Genetics, Inc., Huntsville, AL) was analyzed by PCR with the following pair of primers from human MMP-23 cDNA sequences: 5'-CTTCAGCTTCCGCGAGGTGG-3' (forward primer) and 5'-CTGTCGTCGAAGTGGATGCCG-3' (reverse primer), which correspond to nucleotides 430–449 and 583–663, respectively. The PCR conditions were 30 cycles at 98 C for 10 sec, 68 C for 5 sec, and 72 C for 1 min using Pfu DNA polymerase. The PCR product specifically amplified under these conditions was 241 bp. The result was submitted to the Radiation Hybrid Server of Stanford Human Genome Center (http://shgc-www.stanford.edu) to calculate linkage of the MMP-23 gene to reference markers.

Primary Cell Cultures
Granulosa cells were isolated from immature female Sprague Dawley rats primed with 1 mg diethylstilbestrol in 0.1 ml sesame oil once daily for 3 days by a modification of previously published techniques (38, 39). Briefly, the excised ovaries were suspended in McCoy’s 5A medium (modified) supplemented with 25 mM HEPES, 0.22% NaHCO3, 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 IU/ml penicillin (M5A-H media) containing 6.8 mM EGTA. Ovaries were punctured with a 27-gauge needle, and then incubated for 8 min at 37 C to disrupt the intracellular gap junctions. The released granulosa cells and ovaries were centrifuged and incubated for an additional 4 min with 0.5 M sucrose and 1.8 mM EGTA in M5A-H. Ovarian tissues were gently pressed through 42-mesh stainless steel grid, washed three times, and resuspended in M5A-H. Cell viability was determined by trypan blue exclusion and was normally 70–80%. Viable cells (3x105) were pipetted onto collagen-coated 24-well culture dishes (IWAKI, Tokyo, Japan) in a total volume of 0.5 ml of M5A-H supplemented with 1xITS-x (10 mg/liter of insulin, 5.5 mg/liter of transferrin, 6.7 µg/liter of sodium selenite, and 2 mg/liter of ethanolamine).

Theca-interstitial cells were prepared from immature intact female Sprague Dawley rats (day 24 of age) by the modified procedure originally described by Magoffin (34). Each of the ovaries were cut into four to six pieces, washed twice with 10 ml of Hank’s balanced MEM supplemented with 25 mM HEPES, 0.035% NaHCO3, 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 IU/ml penicillin (H-MEM). Pieces of ovaries were incubated for 60 min at 37 C in 0.25 ml per ovary of collagenase A-DNase I solution including 2.5 mg/ml collagenase A (Roche Molecular Biochemicals) and 100 µg/ml DNase I (Roche Molecular Biochemicals) in H-MEM. The incubated ovaries were flushed every 30 min by gently pipetting through a Pasteur pipette. The dispersed cells were washed three times with H-MEM and passed through 100-µm pore sizes of cell strainers (Becton Dickinson Labware, Lincoln Park, NJ). Theca-interstitial cells were then purified by a discontinuous density centrifugation procedure. Six milliliters of 44% Percoll in H-MEM were carefully layered on top of 56% Percoll cushion in 17 x 100-mm sterile polystyrene Falcon tubes. Dispersed cells (10~25 x 106 cells in 1.5 ml) were layered on top of a 42% Percoll solution and centrifuged at 400 x g for 20 min at 4 C. After centrifugation, the theca-interstitial cells was settled down into 44% Percoll layer above the interface with 56% Percoll cushion. The purified theca-interstitial cells phase was aspirated and then washed three times with M5A-H media. The cells were 90% viable, as determined by trypan blue exclusion. Viable cells (2x105) were placed onto collagen-coated 24-well culture dishes in a volume of 0.5 ml of M5A-H supplemented with 1 x ITS-x. At selected times during culture at 37 C in a humidified 95% air, 5% CO2 environment, medium was removed, centrifuged, and boiled for 10 min. After centrifugation, supernatant was frozen at -80 C until assayed for steroid hormones and cAMP by ELISA.

Progesterone, androstenedione, and cAMP were measured with Progesterone EIA Kit (Cayman Chemical Company, Ann Arbor, MI), Androstenedione ELISA Kit (Oxford Biomedical Research, Inc., Oxford, MI) and cAMP EIA System (Amersham Pharmacia Biotech), respectively, according to manufacturer’s instructions.

In Situ Hybridization
Immature female rats were decapitated, and ovaries and uteri were quickly removed by dissection at the indicated times. Tissues were embedded in Tissue-Tec OCT compound (Miles Laboratories, Kankakee, IL) and frozen in an isopentane-dry ice bath. Sections of 10 µm (ovaries) and 12 µm (uteri) in thickness were cut on a cryostat and mounted on silane-coated microscope slides. Hybridization was performed with digoxigenin-labeled riboprobes synthesized using a DIG RNA Labeling Kit (Roche Molecular Biochemicals) under the slightly modified conditions as described by Schaeren-Wiemers and Gerfin-Moser (35). Briefly, sections were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature and acetylated for 10 min with buffer containing 0.1 M triethanolamine and 0.25% acetate anhydride. Prehybridization was performed at room temperature with 200 µl of hybridization buffer: 50% formamide, 5 x SSC, 5 x Denhardt’s (Wako Pure Chemical Industries Ltd, Osaka, Japan), 500 µg/ml tRNA (Roche Molecular Biochemicals) per slide for overnight. Hybridization buffer containing heat-denatured DIG-cRNA probe was spread over the sections and covered with siliconized coverslips and sealed with DPX mounting reagent (Fluka Chemical Co., Ronkonkoma, NY). The hybridization was performed overnight at 72 C. Slides were washed in 0.2 x SSC at 72 C for 60 min and incubated for 1 h with an anti-DIG antibody (Roche Molecular Biochemicals). Visualization of the signal was performed with nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate solution containing 0.24 mg/ml levamisole for 3 days.

To examine the expression and localization of MMP-23 mRNA in cultured granulosa cells and theca-interstitial cells, 1–2 x 105 cells were each cultured for 48 h on the collagen-coated glass eight-well chamber slides (Becton Dickinson Labware) in 0.5 ml of McCoy’s 5a medium containing 1 x ITS-x with or without stimuli. The cultured cells were fixed in 4% paraformaldehyde in PBS. After fixation and acetylation, the slides were incubated in 1 x SSC for 5 min and permeabilized with 0.2 N hydroxyl chloride for 10 min. In situ hybridization was performed as described above.

Semiquantitative Multiplex RT-PCR
Total RNA was prepared from cultured rat granulosa cells and theca-interstitial cells by ISOGEN (Nippon Gene, Tokyo, Japan). One microgram of total RNA was reverse transcribed with random hexamer by using ThermoScript RT-PCR System (Life Technologies, Inc., Gaithersburg, MD). Specific primers were used to amplify rat MMP-23 cDNA: sense, 5'-CAGGATTCTCTCCTTTCCCC-3'; antisense, 5'-GCCTCTTCATGAGCCTCTGG-3'. Each reaction contained the template DNA corresponding to cDNA synthesized from 50 ng of total RNA, specific primer sets for rat MMP-23, and Quantum RNA alternate 18S internal standards (Ambion, Inc., Austin, TX), which was used as an internal control at the 1:9 ratio of 18S primers to competimers. Multiplex PCR reaction was carried out in 50 µl reaction volumes using Pfu Taq polymerase with 30 cycles of denaturing (97 C, 30 sec), annealing (60 C, 30 sec), and extension (72 C, 2 min). Aliquots of PCR product (5 µl) were electrophoresed through 2% agarose 21 gels (Nippon Gene) in 0.5xTBE. The separated DNA bands were stained with SYBR Green I (Molecular Probes, Inc., Eugene, OR), visualized, and analyzed by computerized densitometric scanning of the images using a lumino image analyzer LAS-1000 (Fuji Photo Film Co., Ltd., Tokyo, Japan). MMP-23 expression was normalized to expression of 18S ribosomal RNA presented as the ratio of fluorescence intensity in the MMP-23 and 18S bands.

Immunological Characterization of MMP-23
Antihuman MMP-23 polyclonal antibodies were raised against a synthetic peptide (329KGKVYWYKDQEPLE342) corresponding to amino acids 329–342 in the carboxyl-terminal domain of human MMP-23, whose sequence is encoded at amino acids 331–344 in rat ortholog. BLAST searching at the National Center for Biotechnology Information web site provided no peptide with greater than 45% level of identity except human, rat, and mouse MMP-23. Two rabbits were immunized with the peptides coupled to keyhole limpet hemocyanin (Sigma Genosys Japan, Hokkaido, Japan). Specificity of the obtained antiserum GN2062 was determined by Western blotting analysis of the membrane fractions prepared from COS-1 cells expressing epitope-tagged human or rat MMP-23. A 1:1,000 dilution of GN2062 specifically recognized both human and rat MMP-23 (see Fig. 8Go). The freshly obtained ovaries were embedded in Tissue-Tec OCT compound and frozen in an isopentane-dry ice bath. Tissue sections of 10 µm thickness were cut on a cryostat and mounted on silane-coated microscope slides. The sections were fixed in 4% periodate-lysine-paraformaldehyde for 10 min at 4 C, washed in PBS, and incubated for 10 min at 90 C with 0.01 M citrate buffer (pH 6.0). After cooling to room temperature, the sections were quenched twice with 50 mM ammonium chloride-PBS for 10 min. Blocking was performed with 10% BSA in PBS containing 0.05% Tween 20 (Roche Molecular Biochemicals) for 30 min at room temperature. Each of the sections was incubated overnight at 4 C with the primary antibody GN2062 (1:200) in PBS containing 0.05% Tween 20 and 1% BSA. Subsequently, sections were washed with PBS containing additional 0.5 M NaCl and 0.1% Tween 20, and endogenous peroxidase activity was blocked by the treatment with 0.3% hydrogen peroxide and 0.3% NaN3 in PBS for 10 min at room temperature. After reacting with peroxidase polymer-conjugated secondary antibody Envision (DAKO Corp., Carpinteria, CA) for 30 min at room temperature, immunoreactive signals were visualized with a mixture of 1 mg/ml diaminobenzidine, 0.65 mg/ml NaN3, and 0.016% hydrogen peroxide. Sections were counterstained with hematoxylin. Specificity of staining was determined by comparing the serial sections incubated with an equal protein amount of nonimmunized rabbit antiserum or with a specific antiserum neutralized by preabsorbing an antiserum overnight at 4 C with a 20-fold (by weight) excess of the synthesized peptide, which was used as the immunogen.


    ACKNOWLEDGMENTS
 
We wish to express our gratitude to Drs. Kevin J. Catt and Gilles Michel for critically reading this manuscript and for encouragement; and to Dr. Duanqing Pei for providing useful information about CA-MMP and helpful discussion.


    FOOTNOTES
 
Address requests for reprints to: Junji Ohnishi, Ph.D, Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan. E-mail: johnishi{at}sci.hokudai.ac.jp

This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education and Culture, Japan, and the Japan Society for the Promotion of Science, and by Research grants from the Nissan Science Foundation and the Kurata Foundation. E. Ohnishi, H.M. and A.K. are supported by Research Fellowships of the Japan Society for the Promotion of Science.

1 Data deposition: The sequences of MIFR reported in this paper have been deposited in the GenBank database (accession numbers, AB010960, AB010961, and AB031068). Back

Received for publication March 3, 2000. Revision received February 14, 2001. Accepted for publication February 20, 2001.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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