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BOR - Papers in Press, published online ahead of print March 9, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.036145
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BIOLOGY OF REPRODUCTION 73, 80–87 (2005)
DOI: 10.1095/biolreprod.104.036145
© 2005 by the Society for the Study of Reproduction, Inc.

Expression of Basigin, an Inducer of Matrix Metalloproteinases, in the Rat Ovary1

A. McDonnel Smedts 2 , and T.E. Curry, Jr. 

Department of Obstetrics and Gynecology, University of Kentucky College of Medicine, Lexington, Kentucky 40536


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The extensive tissue remodeling that occurs during follicular development, ovulatory rupture, and the formation and regression of the corpus luteum (CL) requires local degradation of the extracellular environment by matrix metalloproteinases (MMPs). This report characterizes the expression pattern of basigin (Bsg), a putative regulator of MMP induction, in the rat ovary. An induced superovulation model (eCG/hCG) was used in immature rats to evaluate Bsg expression profiles in ovaries collected during the follicular phase, the preovulatory period, and the luteal lifespan. Levels of Bsg mRNA were unchanged through follicular growth (0–48 h post-eCG) and increased during postovulatory luteinization (24 and 48 h post-hCG; P < 0.01). Bsg expression persisted into pseudopregnancy (4–8 days post-hCG) and after functional luteal regression (12 days post-hCG). The profile of Bsg expression during regression of the CL was examined using a model of induced luteolysis. Both functional and structural regression was associated with a decline in Bsg expression levels. Bsg mRNA and protein localized to the theca of preovulatory follicles (12 h post-hCG) and formative and functional CL (24 h–8 days post-hCG). Bsg expression profiles in the induced ovulation and CL regression models were similar to observations made in naturally cycling mature rats. In the cycling ovary, Bsg signaling localized to newly forming CL, the theca of preovulatory follicles, and appeared to be lower in CL from previous estrous cycles. A putative regulatory mechanism of Bsg expression was identified using an in vitro model; treatment of cultured granulosa cells with hCG significantly augmented Bsg mRNA expression levels. The processes of ovulation and luteogenesis may be facilitated by Bsg expression and its induction or regulation of the MMPs.

corpus luteum, follicular development, ovary, ovulation, theca cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The processes of follicular development, ovulatory rupture, and formation or regression of the CL require tightly coordinated remodeling of the extracellular matrix by the MMPs. Members of the MMP family are structurally related, zinc-dependent proteolytic enzymes distinguished by substrate specificity, regulatory mechanisms, mode of action, and localization. In the ovary, functional regulation of the MMPs relies on a balance of inductive and inhibitory factors [1–3]. Down-regulation of MMP activity is mediated by the tissue inhibitors of metalloproteinases [2], whereas proposed stimulatory factors include gonadotropins, steroid hormones, and tumor necrosis factor {alpha} [4–7]. Additionally, MMPs are induced in normal and neoplastic tissues by a transmembrane glycoprotein of the immunoglobulin superfamily BSG (also known as CD147 and EMMPRIN) [8–12].

BSG is produced by cells of the nervous, immune, and reproductive systems and by several types of cancers [13– 15]. Despite the characterization of the gene and promoter region, the regulatory mechanisms of Bsg gene expression and its function remain ambiguous [16, 17]. Functional studies have ascribed to it numerous roles, including an involvement in neuronal signaling, tumor metastasis, monocarboxylic acid transport, and fertility [14]. Bsg knockout mice display learning and memory disorders, odor insensitivity, and myopia [18–20]. Reproductive deficiencies in the knockout model arise from disrupted spermatogenesis and embryo implantation [21].

Expression of Bsg in the ovary has received limited attention. In female knockout mice, ovarian morphology and ovulatory response appear normal [21]. Kuno et al. describe the expression of Bsg mRNA as limited to cumulus granulosa cells and scarcely detectable in the CL and ovarian stroma in wild-type mice [22]. Conversely, Chang et al. report high Bsg expression in early CL (Days 1–3) and subsequent loss of expression from Days 6 to 15 after hCG stimulation in Kunming White outbred mice [23]. Results of our concurrent research in the rat model (described herein) contrast significantly with patterns characterized in the mouse.

We hypothesized that BSG may be involved in MMP expression and/or activation during normal ovarian dynamics. The initial objective of this research was to characterize the spatial and temporal expression pattern of Bsg in the rat ovary during induced ovulation and CL regression and in the naturally cycling adult rat. Subsequent studies addressed the regulation of Bsg expression in cultured granulosa cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experiments were performed with the approval of the University of Kentucky Institutional Animal Care and Use Committee. Reagents were purchased from Sigma (St. Louis, MO) unless otherwise indicated.

Animal Models

Synchronized folliculogenesis was initiated in prepuberal (21-day-old) Sprague-Dawley rats by administration of 10 IU of eCG followed 48 h later by 10 IU of hCG to induce ovulation and pseudopregnancy (PSP). In this model, ovulatory rupture occurs 12–16 h after hCG injection [24]. Ovaries were collected during follicular development (0, 24, and 48 h after eCG) and during the periovulatory period (12, 24, and 48 h after hCG; n = 3–6 animals per time point). To evaluate Bsg expression in the CL of PSP, we collected ovaries (n = 3 animals per time point) representative of luteal function (peak progesterone production, 4 and 8 days after hCG) and after functional luteal regression (12 days after hCG) as previously established [25]. Expression of ovarian Bsg across the estrous cycle was evaluated in mature animals that exhibited normal cyclicity (>3 consecutive cycles as determined by vaginal lavage); ovaries were collected and snap frozen at 1000 h on the day of estrus, metestrus, diestrus, and proestrus (n = 3 animals per time point) [26]. One ovary from each animal was immediately snap frozen for use in Northern blotting; the second ovary was mounted in OCT compound (VWR, West Chester, PA) for localization studies.

Bsg expression was also assessed during luteal regression using a model of induced functional and subsequent structural luteolysis based on the ablation or replacement of prolactin (PRL) [27, 28]. Ovulation and PSP were induced in immature (21-day-old) rats as previously described (10 IU of eCG followed 48 h later by 10 IU of hCG). Starting on Day 4 of PSP (PSP4) through PSP10, rats were injected twice daily (0800 and 1800 h) with 2-bromo-{alpha}-ergocryptine (bromocriptine) to block endogenous PRL release and induce functional luteolysis (0.5 mg subcutaneously in saline with 0.3% tartaric acid and 10% ethanol). Control animals (n = 4) received vehicle. To induce structural luteolysis, PRL (10 IU) was injected concomitantly with bromocriptine treatment from PSP7 (1800 h) to PSP10 (1800 h); control animals received bromocriptine and the PRL vehicle (saline). Five experimental groups (n = 4) were thus defined: control animals (with functional CL) collected in the morning of PSP4; animals exposed to bromocriptine or vehicle from PSP4 (0800 h) to PSP7 (1800 h) and collected at PSP7 (functionally regressed or functional CL, respectively); and animals injected with bromocriptine from 0800 h PSP4 to 1800 h PSP10 and PRL or vehicle (saline) from 1800 h PSP7 to 1800 h PSP10 and collected at PSP10 (functionally and structurally regressed CL or functionally regressed, structurally normal CL, respectively). At experimental end points (0800 h PSP4, 1800 h PSP7, and 1800 h PSP10), serum samples were collected and both ovaries were removed and weighed. One ovary from each animal was snap frozen; the contralateral ovary was mounted in OCT for in situ hybridization and immunohistochemical analysis.

Northern Blot Analysis of Bsg mRNA Expression

Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to generate a 634-base pair (bp) rat Bsg cDNA fragment. Total RNA (1 µg) isolated from rat preovulatory ovaries (12 h post-hCG), was reverse transcribed using SuperScript II and Oligo dT primers (42°C, 1 h; Invitrogen, Carlsbad, CA). First-strand cDNA samples were amplified using oligonucleotide primers (5'-CACTTGGGCTGGGATAAGAA-3', 5'-CACTTGGGCTGGGATAAGAA-3') based on the reported sequence of rat Bsg (Genebank accession No. NM_012783 ). The PCR conditions included a preincubation period (94°C, 5 min) before addition of Taq polymerase, then 35 amplification cycles (94°C, 30 sec; 58°C, 30 sec; and 72°C, 30 sec). The appropriately sized PCR product was cloned into the pCRII-TOPO Vector (Invitrogen) and sequenced using a Standard ABI kit (Macromolecular Structure Analysis Facility, University of Kentucky, Lexington, KY) to verify that the sequence of the insert corresponded to rat Bsg.

Total RNA was extracted from one ovary per animal by TRIzol (Invitrogen) and quantified by spectrophotometry. Two micrograms of total RNA was subjected to electrophoresis on a 1% agarose-formaldehyde denaturing gel, transferred to a nylon membrane (0.2 µm pore size, Nytran N, Schleicher & Schuell Inc., Keene, NH), and cross-linked to the membrane by baking for 2 h in a vacuum oven at 80°C. Plasmids that contained rat Bsg cDNAs and mouse cDNA for ribosomal protein L32 (kindly provided by Dr. O. K. Park-Sarge, University of Kentucky) were linearized with EcoNI and EcoRI, respectively. 32P-labeled antisense rat Bsg RNA (1 x 106 cpm/µl) and L32 probes were generated using [{alpha}-32P] UTP (10 mCi/ml; NEN, PerkinElmer Life and Analytical Sciences, Inc., Boston, MA) and SP6 or T7 RNA polymerase, as appropriate. Hybridization was performed in NorthernMax buffer (Ambion, Austin, TX) for 12 h at 65°C [29]. Relative band intensities (compared with L32 mRNA expression) were quantitated by PhosphorImaging technology (Molecular Dynamics, Sunnyvale, CA).

Localization of Bsg mRNA by In Situ Hybridization

In situ hybridization was performed as described previously [30]. One ovary from each of three animals was evaluated by in situ hybridization, and at least six sections per ovary were analyzed with the antisense Bsg probe (≥18 tissue sections analyzed per time point). Briefly, frozen sections (10 µm) were fixed in 4% paraformaldehyde and PBS solution, acetylated, and dehydrated in preparation for hybridization. Slides were hybridized overnight with 1 x 106 cpm 35S-labeled antisense rat Bsg RNA probe per slide in a humidified chamber at 55°C. Negative controls were performed with sense rat Bsg RNA probe. Following overnight hybridization, unbound probe was removed by RNase treatment and stringent washes, and slides were processed for autoradiography using Kodak NTB2 emulsion (Eastman Kodak Co., Rochester, NY). After a 3-wk exposure period (4°C), visualization was achieved by development in Kodak D19 and hematoxylin counterstain. Tissues were examined with a Nikon Eclipse E800 microscope (Nikon, Melville, NY) under bright field and dark field optics [26].

Immunohistochemical Localization of BSG

Frozen ovarian sections (10 µm) were fixed in 4% paraformaldehyde solution and blocked with 5% BSA before incubation (4°C overnight) with polyclonal goat anti-rat BSG (1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Bound antibody was labeled with a streptavidin-alkaline phosphatase-conjugated donkey anti-goat secondary antibody (1:1000, 1 h at room temperature; Santa Cruz Biotechnology) and detected using 3-amino-9-ethylcarbazole and light microscopy. Control slides were incubated with preimmune goat serum or with primary antibody that had been preincubated (1 h at room temperature) with an excess (5-fold by weight) blocking peptide (Santa Cruz Biotechnology).

Granulosa Cell Culture

Granulosa cells were collected by needle puncture from the ovaries of immature rats treated 48 h previously with 10 IU of eCG to initiate follicular growth [31]. Oocytes were excluded from collection by mesh filtration (70 µm). The pooled, eCG-primed granulosa cells were plated into 6-well plates (1 x 106 cells/ml) in serum-free D-MEM/F-12 media (Invitrogen; 5% w/v BSA, 1% v/v insulin-transferrin-sodium selenite media supplement) with treatment or control vehicle and incubated 6, 12, 24, or 48 h at 37°C with 5% CO2. After treatment with 1 IU/ml of hCG or control media (n = 3–6 wells per treatment), cells were collected by scraping and centrifugation. Total RNA was collected for analysis by Northern blot as described herein.

Progesterone Assay

Samples (conditioned granulosa cell medium or serum) were diluted as necessary and assayed by sequential competitive immunoassay according to the manufacturer's instructions (IMMULITE; Diagnostic Products Corporation, Los Angeles, CA). The analytical sensitivity of the kit was 0.2 ng/ml.

In Situ Detection of DNA Fragmentation and Apoptosis

Apoptotic cells were identified in ovarian sections in situ using the ApopTag Plus Fluorescein Detection kit (Chemicon, Temecula, CA) according to the directions of the manufacturer. Briefly, digoxigenin-labeled nucleotides were catalytically linked to 3'-OH DNA termini by terminal deoxynucleotidyl transferase (TdT). Incorporated (digoxigenin-labeled) nucleotide oligomers were localized with a fluorescein-conjugated, anti-digoxigenin antibody. Tissues were then counterstained with propidium iodide. Negative controls were performed in the absence of TdT; slides pretreated with DNase I (1 U/µl, RT, 10 min) served as positive controls. Standard fluorescence microscopy was used to visualize apoptotic cells. Labeled cells were counted (under a magnification of 100x) in three randomly selected fields in three CL per ovary (one ovary per animal; four animals per group).

Statistical Analysis

Graphic data were presented as the mean ± SEM. Discrete group means were compared by one-way analysis of variance; post hoc pairwise comparisons were made using the protected least significant difference test. Differences were considered statistically significant at P < 0.05. Homogeneity of variance was confirmed by the Bartlett test during statistical analysis; P ≥ 0.05 was considered acceptable.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The results of our studies demonstrate novel ovarian expression profiles of the putative MMP-inducer Bsg during the rat estrous cycle. Bsg mRNA was detected by Northern blot in untreated, immature ovaries (0 h post-eCG), and levels remained constant through induced follicular maturation (24 h [data not shown] and 48 h after eCG and 0 h after hCG). Expression increased during the preovulatory period (12 h after hCG) and with postovulatory luteinization (24 and 48 h post-hCG; P < 0.01; Fig. 1A). Bsg was expressed over the lifespan of the CL (4–8 days post-hCG), and expression was maintained through functional luteal regression (12 days post-hCG; P < 0.05; Fig. 1B).



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  		FIG. 1. Northern blot analysis of Bsg transcript in total RNA extracted from ovaries collected from eCG-primed rats (n = 3–6) at various time points after hCG administration. A) Bsg mRNA expression (relative to levels measured at 0 h post-eCG) during the periovulatory (0–24 h post-hCG) and early luteal (24–48 h post-hCG) periods (P < 0.01). B) Bsg mRNA expression (relative to levels measured at 48 h post-hCG) during induced PSP. Means ± SEMs are plotted. Dissimilar superscripts denote significant differences (P < 0.05)

Analysis by in situ hybridization revealed Bsg mRNA in thecal and stromal compartments during follicular growth (Fig 2B). Granulosal expression was apparent during early follicular development (0 and 24 h post-eCG) but appeared diminished in follicles immediately preceding ovulation (12 h post-hCG). A notable increase in labeling intensity was observed in the theca that surrounded the preovulatory follicles (12 h post-hCG), in luteinizing granulosa cells (24 h post-hCG), and in newly formed CL (48 h post-hCG; Fig. 2B). During follicular growth (0 h post-eCG and 0 h post-hCG), extensive immunolabeling was apparent in thecal and stromal compartments and the granulosa cells of most healthy follicles. Cumulus granulosa cells expressed minimal levels of Bsg, and oocytes lacked evident labeling by in situ hybridization and immunohistochemical analysis. Specificity of immunoreactivity was verified by the use of primary antibody that had been preincubated with an excess of blocking peptide, which resulted in the absence of reaction product (Fig. 2C).



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  		FIG. 2. Expression of Bsg localized by in situ hybridization (ISH) and immunohistochemistry (IHC) during induced follicular growth and ovulation. A) ISH bright field and (B) ISH dark field images (original magnification x80). C) BSG immunolabeling (original magnification x200). AF, Atretic follicle; F, follicle; GC, granulosa cells; lGC, luteinizing granulosa cells; nCL, new corpus luteum; O, oocyte; pF, preovulatory follicle; S, stroma; Th, theca layer

Expression of Bsg mRNA persisted in luteal tissue through the functional lifespan of the CL (4 and 8 days post-hCG) and was readily detectable in CL after functional regression (12 days post-hCG; Fig. 3). Luteal labeling appeared uniform across the CL with the exception of the cells and connective tissue of the centrum (Figs. 3 and 4). Although the intensity of Bsg mRNA labeling was greatest in luteal tissue, labeling was also detectable in stromal and thecal tissue and in granulosa cells of some small follicles.



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  		FIG. 3. Luteal expression of Bsg localized by in situ hybridization (ISH) over the lifespan of the CL during induced PSP. A) Bright field and (B) dark field images (original magnification x40). C, Centrum of the CL; F, follicle; nCL, new corpus luteum; S, stroma; Th, theca



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  		FIG. 4. Bsg mRNA in ovaries of naturally cycling mature rats. A) Bright field image and (B) Bsg mRNA localization by in situ hybridization (dark field) (original magnification x100 for proestrus and x40 for estrus, metestrus, and diestrus). C) Immunolocalization of BSG (original magnification x200). F, follicle; C, centrum of the CL; nCL, new corpus luteum; ov, oviduct; pCL, corpus luteum from previous cycle(s); Th, theca

To confirm that the expression profile we characterized in the induced ovulation model was representative of Bsg expression during normal (i.e., unstimulated) ovarian function, we evaluated Bsg expression in ovaries collected daily during the 4-day estrous cycle from mature, naturally cycling rats. Results of our analysis demonstrated corresponding patterns of Bsg expression between the induced and naturally ovulating models. For example, in situ hybridization signals for Bsg mRNA in the cycling ovary were strongest in the theca of preovulatory follicles (proestrus) and in newly forming CL of estrus but were also apparent in CL from previous cycles (Fig. 4B). Similarly, BSG protein was localized to the thecal cells of preovulatory follicles on the day of proestrus and to the theca, stroma, and formative CL on the day of estrus. Both in situ hybridization and immunohistochemical analysis demonstrated Bsg expression in new CL and CL of previous cycles during metestrus and diestrus (Fig. 4C).

Parameters of luteal function and structural integrity and Bsg expression as measured in the induced regression model are represented in Figure 5. Three days of bromocriptine treatment (from PSP4 to PSP7) resulted in the functional regression of CL in PSP animals as evidenced by the sharp decline in serum progesterone (Fig. 5A) and a decrease in ovarian weight (Fig. 5B). Ovaries of vehicle-treated animals maintained function (i.e., progesterone production); ovarian wet weights were not statistically different from PSP4 control ovaries. Functional regression was associated with an increase in the number of apoptotic cells detected in luteal sections (Fig. 5C). Ovaries exposed to replacement PRL (from PSP7 to PSP10) after 3 days of bromocriptine treatment underwent structural regression subsequent to functional regression as evidenced by decreased progesterone production, reduced wet weight, and a significantly increased number of apoptotic CL cells (Fig. 5A–C). Bsg expression, measured by Northern blotting, was reduced (P < 0.05) in ovaries with functionally regressed CL; levels were further decreased in structurally regressing ovaries (Fig. 5D). Localization studies (immunohistochemistry and in situ hybridization) confirmed the expression of Bsg in the functionally and structurally regressing CL at all time points analyzed (data not shown).



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  		FIG. 5. Parameters of luteal function, structural integrity, and Bsg expression in the PRL ablation-replacement model of induced luteal regression. Group treatments are indicated across the abscissa; ovaries collected on PSP4 were fully functional, ovaries collected on PSP7 were functionally normal or functionally regressed (without or with bromocriptine treatment, respectively), and ovaries collected on PSP10 were functionally regressed (bromocriptine without PRL replacement) or functionally and structurally regressed (bromocriptine with PRL replacement). A) Serum progesterone (log[progesterone ng/ml]). B) Ovarian wet weight. C) Numbers of apoptotic cells in luteal sections as detected by anti-digoxigenin (fluorescein) labeling. D) Northern blot analysis of Bsg mRNA expression relative to L32. Means ± SEMs are plotted. Dissimilar superscripts denote significant differences (P < 0.05; n = 4)

The elevated levels of Bsg mRNA in luteinizing granulosa cells prompted us to test its regulation by a luteotropic hormone, hCG. Progesterone production by cultured granulosa cells was induced by hCG as measured in conditioned media at 12, 24, and 48 h of culture; levels of progesterone in the media of control groups remained negligible across time points (Fig. 6A). Bsg mRNA was readily detectable in untreated granulosa cells cultured for 0, 6, 12, 24, and 48 h. Addition of 1 IU/ml of hCG to the culture media accelerated the expression of Bsg mRNA compared with control cells. Bsg expression was significantly elevated in gonadotropin-stimulated cells by 12 h of culture but not in control cells until 24 h (P < 0.05). By 48 h of culture, control and treated cells expressed approximately equivalent levels of Bsg mRNA (Fig. 6B).



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  		FIG. 6. Progesterone concentrations in the conditioned media (A) and Bsg mRNA expression (B) of granulosa cells cultured in vitro without (open bars) and with (solid bars) hCG treatment. Treatment means ± SEMs are plotted. Dissimilar superscripts denote significant differences between treatments (control vs. hCG) within each time point and differences over time within a treatment (P < 0.05; n = 3–6)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Results of these experiments indicate for the first time that Bsg is differentially expressed in the rat ovary during follicular development, ovulatory rupture, and CL formation and regression. Distinctive spatial and temporal patterns were observed during an estrous cycle. During follicular growth (0 h post-eCG–0 h post-hCG), a period of significant structural reorganization, Bsg was expressed in granulosal, thecal, and stromal compartments of the ovary. Thecal Bsg expression was particularly notable immediately before ovulation, when Mmp2 (gelatinase A) and Mmp14 (MT1-MMP) are also highly expressed in these cells [4]. After ovulation, levels of mRNA for BSG in whole ovarian extracts rose, and expression (transcript and protein) was localized to newly forming CL, possibly implicating a role for this glycoprotein in the dynamics of luteinization (e.g., cell differentiation, extracellular matrix remodeling, and angiogenesis). Expression profiles observed in the induced model of ovulation closely paralleled those of naturally cycling rats; in particular, the extensive labeling of Bsg mRNA at 24–48 h post-hCG was similar to that seen in the newly formed CL of metestrus.

Closer examination of the expression of Bsg during functional and structural luteal regression was achieved using an induced model of luteolysis. Although Bsg expression appeared to plateau during functional regression in the induced ovulation model (8–12 days post-hCG), a slight decrease in Bsg transcript was measured in CL induced to functionally regress by ablation of endogenous PRL production (for example, bromocriptine- versus vehicle-treated ovaries collected at PSP7). Structural regression induced by PRL replacement (i.e., PSP10) was associated with a significant decline in Bsg expression, which paralleled our observation of reduced Bsg expression in the CL from previous cycles in naturally cycling animals. The outcome of these studies points to a role for BSG in the development or function of the early CL, although a role in luteolysis cannot be ruled out.

Previous studies have demonstrated that BSG on (or released by) tumor cells induced the production and activity of several MMPs, to include MMP1 (collagenase 1) and MMP2 (gelatinase), by neighboring fibroblasts [10–13, 32]. It is tempting to hypothesize that BSG may have a similar (i.e., MMP-inducing) effect in the formative CL. In fact, mRNA encoding MMP2 and MT1-MMP (a gelatinase activator) is highly abundant in the theca of preovulatory follicles and in the postovulatory, luteinizing follicle. Gelatinolytic activity patterns (assessed by in situ zymography) also correlate spatially and temporally with our characterization of early luteal Bsg expression [25, 30, 33]. The gene encoding MMP13 (Mmp13) is expressed late in the luteal lifespan of PSP (Days 13–21) [34]; we speculate that this increase may be elicited by the elevated expression of Bsg that we observed during luteal regression in the PSP model (i.e., Day 12). Although evidence thus far is limited to correlative expression patterns, a direct involvement of BSG in the regulation of ovarian MMPs seems likely and warrants further investigation.

Functional regulation of BSG appears to occur at several levels. Bsg expression was increased by amphiregulin and epidermal growth factor in breast tumor cells, and posttranscriptional processing (i.e., protein synthesis and glycosylation) in endometrial stromal cells was augmented by progesterone treatment [35, 36]. Administration of phorbol 12-myristate 12-acetate, a diacylglycerol analogue that stimulates the protein kinase C signaling pathway, induced the release of bioactive BSG from the surface of tumor cells via the shedding of microvesicles in a time- and dose-dependent manner [37]. A recent report by Tang et al. describes BSG regulation by a positive feedback loop whereby BSG-induced MMPs cleave BSG from the surface of cells, permitting the soluble molecule to further stimulate the production of MMPs (and BSG) by neighboring cells [38]. Our studies have revealed that Bsg mRNA expression increased during granulosa-luteal differentiation in response to a luteinizing stimulus (i.e., hCG) and during autoluteinization of these cells in culture. Although the induction of progesterone production by hCG in cultured granulosa cells was associated with increased Bsg expression, levels of Bsg mRNA also rose, albeit more gradually, in untreated cells that did not produce significant levels of progesterone (Fig. 6). We suggest that an element of the luteinizing cascade, independent of progesterone action, directs Bsg expression in the rat ovarian follicle and that BSG may thereby regulate the (MMP-driven) extracellular matrix remodeling inherent to luteinization.

The specific mechanism of MMP induction by BSG, however, remains unclear. To initiate cell signaling and elicit its downstream effects, the secreted or membrane-bound protein engages in homophilic interactions on neighboring cells or acts as a ligand for an as yet unidentified receptor [10, 39]. Protein tyrosine kinases appear to be critical in the signaling cascade initiated by BSG as treatment with genistein, a protein kinase inhibitor, ablated BSG-stimulated Mmp13 expression by cultured fibroblasts [40]. Genistein also inhibited Mmp13 expression by whole rat ovaries perfused in vitro with LH [41]; it therefore seems plausible that BSG could regulate ovarian MMP13 via a similar pathway to that described in tumor-stroma interactions. In human lung fibroblasts, BSG stimulated Mmp13 expression through the p38 signaling cascade, whereas induction of Mmp2 was mediated through the phospholipase A2/5-lipoxygenase catalyzed pathway in breast cancer cells [40, 42]. Alternate signaling pathways may serve as a mechanism by which BSG regulates the expression of different MMPs.

Although endometrial Bsg expression has been described in the mouse [22, 43], rat [44], and human [36], ovarian studies have thus far been limited to mice. A Bsg knockout mouse model has been used to demonstrate the critical role for BSG in reproduction. Spermatogenesis in Bsg null males was completely disrupted, and null female infertility was associated with impeded embryo implantation and development [21]. Morphologically, the ovaries of Bsg-null female mice appeared normal, and ovulation could be induced with the standard eCG/hCG protocol. Because sexual behaviors and ovulation rates were unaffected by the loss of Bsg, hormonal imbalance and decreased responsiveness to hormones were excluded as factors that cause the reproductive failures of knockout females [21, 22]. Further studies demonstrated abnormalities in embryonic implantation as the prominent cause of female sterility [22]. In wild-type female mice, Bsg mRNA and immunostaining were observed in the granulosa cells of preantral follicles, oocytes, and newly formed CL [23, 45]. In contrast, the findings of the present study in the rat ovary demonstrate minimal Bsg expression in the granulosa cells of preovulatory follicles and no overt labeling in oocytes. Notably, the thecal layer of preovulatory follicles (12 h post-hCG and on the day of proestrus) labeled intensely for Bsg mRNA and protein in the rat (but not the mouse). High expression was observed in new CL of both species, perhaps reflecting a conserved function for BSG in luteinization. In mice, Bsg expression was limited to the first 3 days of the luteal lifespan [23]; our data in the rat indicate that Bsg mRNA is expressed through the functional (i.e., progesterone-producing) phase of the CL. In ovaries of cycling animals, Bsg mRNA labeling is apparent (albeit less intense) in CL persisting from previous cycles.

We report the novel characterization of Bsg expression patterns in the rat ovary and propose a central role for this glycoprotein in the regulation of MMPs during ovarian function. The complex process of luteinization requires intricately regulated remodeling of the extracellular matrix by MMPs to facilitate tissue restructuring, cell differentiation, and angiogenesis. Likely, BSG participates in the regulation of luteinization as an inducer or activator of the MMPs.


   ACKNOWLEDGMENTS
 
We thank Dr. Romana Nowak (University of Illinois) for her guidance and insight into this project and extend our appreciation to Sarah Wheeler-Price (University of Kentucky) for her assistance with the ApopTag labeling.


   FOOTNOTES
 
1 Supported by National Institutes of Health grant P20RR15592. Back

2 Correspondence: A. McDonnel Smedts, Department of Obstetrics and Gynecology, University of Kentucky Medical Center, 800 Rose St., Lexington, KY 40536. FAX: 859 323 3761; asmed2{at}email.uky.edu Back

Received: 8 September 2004.

First decision: 23 September 2004.

Accepted: 2 March 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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