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Biology of Reproduction 67, 1522-1531 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Expression and Localization of Thrombospondin-1 and -2 and Their Cell-Surface Receptor, CD36, During Rat Follicular Development and Formation of the Corpus Luteum1

Jim J. Petrika, Patricia A. Gentrya, Jean-Jacques Feigeb, and Jonathan LaMarre2,a

a Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph,> Guelph, Ontario, Canada N1G 2W1 b INSERM EMI 0105, DBMS/BRCE, CEA-Grenoble, 38054 Grenoble Cedex 9, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Thrombospondin (TSP)-1 and -2 are extracellular matrix glycoproteins that are both antiangiogenic and important in regulating cellular development, differentiation, and function. To evaluate the expression of TSP in follicular and luteal development, ovarian cycles of Sprague-Dawley rats were synchronized and tissues collected daily at stages corresponding to the early antral, ovulatory, early luteal, and late luteal phases of the ovarian cycle. Immunohistochemistry and Western blot analyses demonstrated that TSP-1 protein and its receptor, CD36, were present in the early antral phase and were localized primarily to the granulosa cells of antral follicles. Both proteins were also present immediately after ovulation and were localized to the developing corpus luteum. Messenger RNA for TSP-1 showed a similar pattern, with expression at the early antral and ovulatory phases. Protein and mRNA expression for TSP-2 was relatively delayed compared to TSP-1, although TSP-2 also was expressed in granulosa cells. Both TSP-1 and -2 were increased in response to LH stimulation in vitro, whereas TSP-2 was suppressed by FSH. The temporal pattern of expression of TSP-1, -2, and CD36, which mirrors the active phases of angiogenesis in this experimental model, is compatible with a role for these proteins in the control of ovarian vascularization.

corpus luteum, follicle, follicular development, ovary, ovulatory cycle


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mammalian ovarian cycle is characterized by the growth of numerous follicles in response to various hormones and local peptide growth factors. Under the control of many different stimuli, the majority of these follicles will undergo atresia, with one or several follicles progressing to ovulation and subsequent formation of the corpus luteum [1]. Three specialized cell types form the layers that line the follicle: the granulosa, the theca interna, and the theca externa. After ovulation, the theca and granulosa cells are remodeled, resulting in a vascular infiltration of the previously avascular granulosa cells [2]. Around this time, the steroidogenic properties of the theca and granulosa cells are altered, causing these cells to differentiate into lutein tissue and to form the corpus luteum. The corpus luteum functions to produce progesterone, which is necessary for maintenance of pregnancy should a pregnancy occur [3]. If the oocyte is not fertilized, regression of the corpus luteum occurs, and a new ovarian cycle is initiated. Some factors involved in the control of this exquisitely choreographed developmental program have been elucidated, but the overall mechanisms of control remain poorly understood. Peptide growth factors, such as insulin-like growth factor (IGF)-I and -II [4], transforming growth factor ß (TGFß) [5], and epidermal growth factor [6], have been shown to participate in the growth and differentiation of cells during folliculogenesis. Factors within the extracellular matrix (ECM) have also been reported to be important in the regulation of cellular function in a number of physiological systems and are potentially important in the control of follicular development and atresia. Included in this group of ECM proteins are members of the thrombospondin (TSP) family, TSP-1 and TSP-2.

Both TSP-1 and -2 are large, trimeric, inducible glycoproteins secreted by several cell types and found in the ECM [7–9]. These two isoforms are similar in structure, but they differ in their location and levels of expression. Both TSP-1 and -2 have been implicated in a number of processes, including adhesion and migration of cells, cellular growth, platelet aggregation, and angiogenesis [10]. Through binding to surface receptors such as CD36, the TSPs act as autocrine growth factors, mediating different aspects of cellular function [11–14]. In modulation of bone cell proliferation and function, TSP has been shown to be colocalized with IGF-I and TGFß in the ECM and subject to steroid regulation similar to that of the local growth factors [15]. Although some controversy has surrounded the issue of whether TSP acts as an inhibitor or an enhancer of angiogenesis (refer to review articles [16] and [17]), it is now relatively widely accepted that TSP-1 and -2 inhibit angiogenesis and cell migration [18–22]. The exact mechanisms by which the TSPs exert their effects on angiogenesis are unclear; however, recent studies have shown that binding to CD36 causes intracellular signaling that initiates apoptosis of endothelial cells, inhibiting neovascularization [23, 24]. This is consistent with observations that targeted TSP-2 inactivation in vivo results in a reduction of tumor cell apoptosis and increased tumor vascularization [25]. In the ovary specifically, isolated rodent granulosa cells have been shown to produce TSP (probably TSP-1), which in turn binds to the cell surface [26]. In these cells, a positive correlation was found between the amount of TSP synthesized and the rate of cell proliferation, suggesting that TSP exerts an autocrine effect on granulosa cell growth and maturation. A more recent study also suggested that human granulosa cells express cellular binding sites for TSP [27], although the nature of these sites has not been characterized.

The effect of TSP-1 and -2 on angiogenesis and granulosa cell growth and differentiation suggests that these important regulatory proteins may be involved in multiple aspects of follicular growth and atresia and in formation of the corpus luteum, yet little is known concerning their expression in these sites. To begin characterizing potential roles for the TSPs during the ovarian cycle, we have examined the expression of TSP-1 and -2 and their receptor, CD36, during folliculogenesis and formation of the corpus luteum in the rat. To further explore the mechanisms underlying the observed cyclical changes in TSP expression, we have also performed in vitro studies with gonadotropins on cultured granulosa cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Tissue Collection

Prepubescent Sprague-Dawley rats (Charles River, Wilmington, MA) were received at Postnatal Day (Day) 19 and allowed to acclimatize for 1 wk before intervention. At Day 26, follicular development and maturation were stimulated with s.c. injection of 25 IU of eCG. Synchronous ovulation was achieved on Day 28 with i.p. injection of 10 IU of hCG. Animals were killed and tissues collected on days representative of early antral (Days 26 and 27), ovulatory (Days 28 and 29), early luteal (Day 30), and late luteal (Days 33, 36, and 40) phases of the ovarian cycle. Ovaries were trimmed of fat and connective tissue. Tissue to be used for immunohistochemistry was fixed in 10% (v/v) neutral buffered formalin (Fisher Scientific, Nepean, ON, Canada) overnight at 4°C and processed according to normal histological procedures. For protein determination, ovaries were dissociated in the presence of lysis buffer (5% [w/v] sodium deoxycholate, 0.1% [w/v] PMSF, 10% [v/v] Nonidet P-40, 0.1% [w/v] aprotinin 5 mg, and 1% [w/v] SDS). Tissues to be used for Northern blot analysis were flash-frozen in liquid nitrogen for RNA extraction.

Tissue Culture

Spontaneously immortalized rat granulosa cells were kindly provided by Dr. Robert Burghardt (Department of Anatomy, Texas A&M University, College Station, TX). Granulosa cells (5 x 105) were plated in 6 ml of Dulbecco modified Eagle medium (DMEM)/F12 (with 2% [v/v] penicillin/streptomycin) supplemented with 10% (v/v) fetal calf serum (Sigma Chemicals, St. Louis, MO) for 12 h to promote attachment. After the initial culture period, the cells were switched to serum-free DMEM/F12 for 24 h. Granulosa cells were subsequently cultured in serum-free media supplemented with either FSH from porcine pituitary extracts (50 ng/ml; Vetrepharm, Belleville, ON, Canada) or LH from human pituitary extracts (100 ng/ml; Sigma) for 6, 12, or 24 h. Control cells were cultured in serum-free media for 12 h. At the end of the culture period, RNA was isolated using Trizol reagent (Gibco BRL, Burlington, ON, Canada) as indicated by the manufacturer.

Immunohistochemistry

Histological sections of ovaries (thickness, 5 µm) were cut from paraffin blocks with a rotary microtome and mounted on glass microscope slides (Superfrost Plus; Fisher). Immunohistochemistry was performed to localize TSP-1, TSP-2, and CD36 in ovaries at various stages of the ovarian cycle using a modification of the avidin-biotin peroxidase method [28]. Briefly, slides were incubated for 24 h at 4°C in a humidified chamber with either mouse anti-human TSP-1 (1:400 [v/v] dilution; Medicorp, Inc., Quebec, QC, Canada), mouse anti-TSP-2 (1:500 [v/v] dilution), or mouse anti-human CD36 (1:300 [v/v] dilution). All antisera were diluted in 0.01 M PBS (pH 7.5) containing 2% (w/v) BSA and 0.01% (w/v) sodium azide (100 µl/slide). All subsequent incubations were at room temperature. Biotinylated horse anti-mouse IgG (1:100 [v/v] dilution; Vector Laboratories, Burlingame, CA) or anti-rabbit IgG (Sigma) was diluted in the same buffer and applied for 2 h. The slides were then washed in PBS and incubated with avidin and biotinylated horseradish peroxidase (1:30 [v/v] dilution, Extravidin; Sigma). Peptide immunoreactivity was localized by incubation in fresh diaminobenzidine tetrahydrochloride (DAB tablets, 10 mg; Sigma) with 0.03% (v/v) hydrogen peroxide for 2 min. Tissue sections were counterstained with Carazzi hematoxylin for 1 min. Tissues were dehydrated and placed under coverslips with Permount (Fisher). To establish specificity of staining, primary antisera were substituted with nonimmune serum. For additional controls, the staining procedure was performed in the absence of secondary antibody. In each case, staining was abolished.

Western Blot Analysis

Thrombospondin-1, -2, and CD36 protein levels were detected and quantified using Western blot analysis. Twenty micrograms of total protein extracted from rat ovaries were subjected to SDS-PAGE electrophoresis using an 8% (v/v) separating gel. The separated proteins were electrotransferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA). Membranes were blocked overnight with 5% (w/v) skim milk at 4°C. Membranes were then incubated for 2 h at room temperature with either mouse anti-human TSP-1 (1:500 dilution; Medicorp), mouse anti-human TSP-2 (1:300 dilution), or mouse anti-human CD36 (1:400 dilution) on a rocking platform. After washing with TBS (Tris-buffered saline) and 1% (v/v) Tween 20 and reblocking, peroxidase-conjugated sheep anti-mouse (1:800 dilution) was added for 1 h at room temperature on a rocking platform. Antibody binding was detected with enhanced chemiluminescence (Boehringer Mannheim, Laval, QC, Canada) and Fuji medical x-ray film (Konica Medical Imaging, Wayne, NJ).

Northern Blot Analysis

Northern blot analysis was performed on RNA from freshly isolated ovaries and from in vitro-cultured, spontaneously immortalized rat granulosa cells. Ovaries were removed and flash-frozen as described above. Tissues were then immersed in cold Trizol reagent and homogenized with a Brinkman PT3000 polytron (Fisher Scientific). Total ovarian RNA was isolated according to the manufacturer's instructions. Twenty micrograms of total cellular RNA from each day of collection was subjected to electrophoresis on an 0.8% (w/v) formaldehyde/agarose gel. Following separation, RNA was transferred to a nylon membrane (Hybond-N; Amersham Pharmacia Biotech, Buckinghamshire, U.K.) using passive capillary transfer. Membranes were ultraviolet cross-linked and hybridized for a minimum of 2 h in prehybridization buffer (6x SSPE [1x SSPE: 150 mM NaCL, 10 mM NaH2PO4, and 1 mM Na/EDTA], 0.5% SDS, 5x Denhardt reagent, 2 µg of salmon sperm DNA, and 50% [w/v] formamide). The cDNA probes for murine TSP-1 and -2 (kindly provided by Dr. P. Bornstein, Department of Biochemistry and Medicine, University of Washington, Seattle, WA) were radiolabeled using [{alpha}32P]dCTP (Amersham Life Sciences, ON, Canada) and Rediprime random primer labeling (Amersham, Piscataway, NJ). The radiolabeled probes were hybridized overnight in the identical hybridization buffer. Membranes were washed twice for 15 min in SSC (1x SSC: 0.15 M sodium chloride and 0.015 M sodium citrate)/1% (w/v) SDS at 42°C and once in 0.1x SSC/0.5% SDS for 20 min at 55°C. Membranes were blotted dry and analyzed in a Molecular Imager GS250 (BioRad, Richmond, CA) located in the Clarice Chalmers Molecular Imaging Facility (Department of Biomedical Sciences, University of Guelph, Guelph, ON, Canada). To determine equivalency of load and transfer, membranes were stripped by washing in 0.01x SSC and 0.5% SDS for 30 min at 90°C and rehybridized with a probe for murine 7S ribosomal RNA.

Statistical Analysis

Densitometric data for Western and Northern blot analyses were subjected to two-way analysis of variance with threshold of significance at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cell-Specific Localization of TSPs and CD36

After synchronization of the ovarian cycle, immunohistochemistry was performed on formalin-fixed, paraffin-embedded ovarian sections to determine expression and localization of TSP-1 and -2 and of the TSP receptor, CD36. On Days 26 and 27, which are representative of the preantral and early antral phase of the cycle, staining for TSP-1 was localized primarily to granulosa cells of the follicles, although some theca interna cells were faintly immunopositive (Fig. 1, A–D). Shortly after ovulation, during the early luteal phase of the ovarian cycle (Day 30), TSP-1 staining was evident in the corpus luteum (Fig. 1, E and F). Throughout the later luteal phase of the cycle (Days 33–40), immunostaining for TSP-1 was not present in the corpus luteum or in the small follicles in the ovary (Fig. 1, G and H).



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  		FIG. 1. Immunohistochemical localization of TSP-1. Immunopositive cells stained brown and demonstrated localization of TSP-1 protein to the ECM of granulosa cells during the preantral and early antral phase of the cycle on Days 26 (A and B) and 27 (C and D). By Day 30 (E and F), during the early luteal phase of the cycle, staining was evident in the corpus luteum (CL). No staining was apparent at Day 40 (G and H), during the late luteal phase of the cycle. Negative controls in which primary antibody was replaced with preimmune serum are also shown (I and J). Similar controls were utilized for all immunohistochemical experiments. Magnification x250 (A, C, E, G, and I) and x1000 (B, D, F, H, and J)

Immunohistochemistry demonstrated a slightly different pattern of expression for TSP-2. No cells were observed to be immunopositive for TSP-2 at Day 26 or 27 (Fig. 2, A–D). However, in a fashion similar to TSP-1, during the early luteal phase of the cycle (Day 30) (Fig. 2, E and F), expression of TSP-2 protein was high and localized to luteal cells within the corpus luteum. By Day 40, during the late luteal phase of the cycle, no cells immunopositive for TSP-2 were present (Fig. 2, G and H).



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  		FIG. 2. Immunohistochemical localization of TSP-2 in rat ovaries at Days 26 (A and B), 27 (C and D), 30 (E and F), and 40 (G and H). No staining was apparent in the ovary during the preantral and early antral phases of the ovarian cycle (Days 26 and 27). During the early luteal phase (Day 30), protein expression was evident in the matrix of the luteal cells of the corpus luteum. Staining was absent by the late luteal stage (Day 40). Magnification x250 (A, C, E, and G) and x1000 (B, D, F, and H)

The immunohistochemical analyses of CD36 expression revealed a staining pattern similar to that of TSP-1. The CD36 antigen was most pronounced in granulosa cells of preantral and early antral follicles on Days 26 and 27 (Fig. 3, A–D). Moderate CD36 staining was also evident in the vascular thecal layers. Once again, after ovulation, CD36 expression was observed homogeneously in luteal cells of the corpus luteum (Fig. 3, E and F). By the late luteal phase, no staining for CD36 was evident in the ovary (Fig. 3, G and H).



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  		FIG. 3. Immunohistochemical localization of CD36. Protein for receptor CD36 was localized to the periphery of granulosa cells during the early antral phase of the cycle on Days 26 (A and B) and 27 (C and D). Receptor antigen was more diffusely distributed around the luteal cells of the corpus luteum on Day 30 (E and F) and was absent in all areas of the ovary by Day 40 (G and H). Magnification x250 (A, C, E, and G) and x1000 (B, D, F, and H)

Throughout the ovarian cycle, some follicles were at various stages of atresia and regression. We noted no difference in staining characteristics for TSP-1, -2, or CD36 in these follicles as compared to healthy follicles, although these data were not quantified (data not shown).

Expression of TSP Ligand and Receptor Protein in the Ovary

The TSP-1 and CD36 antigens were detected concurrently in two peaks, the first occurring at Days 26 and 27 (follicular phase) and the second during a highly defined peak for TSP-1 on Day 30 that extended to Day 33 for CD36, at which time TSP-1 was no longer detected (Fig. 4). The observed differences for both TSP-1 and CD36 antigens were statistically significant (P < 0.05) on Days 26, 27, and 30. In contrast to the dual peaks of expression observed with TSP-1 and CD36, TSP-2 antigen was detected as a single, statistically significant (P < 0.05) peak beginning in the early luteal phase (Day 30) and extending to Day 36 (Fig. 4B). A very slight increase in TSP-2 antigen, however, was observed on Day 28, during the late follicular phase. No expression of TSP-1, -2, or CD36 was observed within the ovarian extracts late in the ovarian cycle (Day 40).



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  		FIG. 4. Western blot analysis for TSP-1 (A), TSP-2 (B), and CD36 (C). Expression of TSP-1 protein was significantly increased on Days 26, 27, and 30. Expression of TSP-2 protein was elevated on Days 30, 33, and 36. The pattern of protein expression for CD36 mirrored that of TSP-1, with a significant increase in protein expression on Days 26, 27, and 30. Graphs indicate the relative densitometry scores for the Western blots. *Difference in densitometric values compared to Day 40 using two-way ANOVA (P < 0.05, n = 4). Open arrows represent s.c. injection of 25 IU eCG on Day 26 and i.p. injection of 10 IU hCG on Day 30

TSP-1 and -2 mRNA Expression Throughout>the Ovarian Cycle

Ovaries from rats at various phases of the ovarian cycle were removed and flash-frozen in liquid nitrogen. Total RNA from these tissues was extracted and subjected to Northern blot analysis using TSP-1 and -2 cDNA probes. The expression pattern of TSP mRNA was consistent with the TSP immunohistochemical staining, and again, TSP-1 and -2 exhibited different patterns of expression throughout the ovarian cycle. The TSP-1 mRNA was expressed during the early antral phase, with significantly increased (P < 0.05) expression at Days 26 and 27 as well as during the early luteal phase on day 30 (Fig. 5). In contrast, TSP-2 mRNA was absent during the early part of the cycle and exhibited a later onset of expression, with a significant increase (P < 0.05) in mRNA on Days 29, 30, and 33 (Fig. 5).



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  		FIG. 5. Northern blot analysis for TSP-1 and -2 mRNA. This representative Northern blot demonstrates distinct patterns of mRNA expression for TSP-1 and -2. TSP-1 was expressed during the preantral and early antral (Days 26 and 27) and early luteal (Day 30) phases of the cycle, whereas TSP-2 expression did not occur until the late follicular developmental (Day 29) and early luteal (Days 30 and 33) phases. The graph represents the relative densitometric analysis of TSP-1 and -2 mRNA in which signal intensity was normalized against 7S RNA. aDifference in TSP-1 expression compared to Day 40 (P < 0.05, two-way ANOVA). bDifference in TSP-2 expression compared to Day 40 (P < 0.05, two-way ANOVA) (n = 4). Open arrows represent s.c. injection of 25 IU eGG on Day 26 and i.p. injection of 10 IU hCG on Day 30

Gonadotropin Control of TSP Expression in Cultured Granulosa Cells

Spontaneously immortalized rat granulosa cells were cultured in the presence of FSH and LH for 6, 12, and 24 h, after which RNA was isolated and subjected to Northern blot analysis (Fig. 6). At all durations of culture, FSH appeared to have minimal effect on TSP-1 expression as compared to nontreated control cells. At 6 and 12 h of culture, FSH suppressed TSP-2 expression, whereas at 24 h of culture, a slight but nonsignificant increase in TSP-2 was observed (Fig. 6). Both TSP-1 and -2, however, were responsive to LH. Treatment with 100 ng of LH for 6 h caused a significant (P < 0.05) increase in TSP-1 expression, and this elevation was increased after 12 and 24 h of culture. The TSP-2 mRNA levels were not changed after 6 h of LH treatment but were significantly (P < 0.05) elevated after 12 and 24 h of culture (Fig. 6).



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  		FIG. 6. Northern blot analysis of in vitro cultures of immortalized granulosa cells treated with FSH and LH. These representative Northern blots demonstrate changes in TSP-1 and -2 mRNA levels in response to 6-, 12-, and 24-h treatment with either 50 ng of FSH or 100 ng of LH. Control cultures involved 12-h, serum-free culture with no treatment. Exposure to LH resulted in an increase in TSP-1 expression at 6, 12, and 24 h and an increase in TSP-2 at 12 and 24 h. Graphs represent the relative densitometric analysis of TSP-1 and -2 in relation to control culture. Data are presented as a percentage of signal from control cultures. The 7S blot is shown to demonstrate equal loading and transfer of RNA. *Difference in TSP-1 or -2 expression compared to control culture (P < 0.05, two-way ANOVA) (n = 3)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The complex tissue processes involved in the mammalian ovarian cycle require numerous highly regulated cell-cell and cell-matrix interactions. The ECM, which is often considered to be simply scaffolding to support resident cells, is actually comprised of numerous glycoproteins, cytokines, and growth factors that mediate intracellular signaling via cell-surface receptors. Among the many important regulatory molecules within the ECM are TSP-1 and -2, which are members of a small family of secreted glycoproteins. The TSPs influence cellular function through direct ligand-receptor interactions with specific cell-surface receptors and also by modulating cell-ECM interactions [29], and they have been included in a list of "matricellular proteins" that facilitate liaisons between structural macromolecules and other factors, such as cytokines, proteases, and growth factors [30]. Through these different activities, the TSPs influence cell adhesion, wound healing, and angiogenesis, all of which are important components of the tissue and cellular changes that occur during the ovarian cycle [31–33].

In mammals, preantral follicles contain avascular granulosa cells and differentiated stromal cells that form the theca [34]. As the follicle develops, vascularization increases, particularly in the thecal layers, and the steroidogenic activity of the follicular cells increases as well [35]. Profound and rapid changes in vascular growth also occur during formation of the corpus luteum [36]. The altered vascularization of cell layers during follicular and luteal development and maturation is particularly important in the context of endocrine function, because it allows rapid cellular proliferation while maintaining functional vascular access to the circulation for secreted hormones. The mechanisms by which changes in vascularization occur are not clearly understood, but many regulatory molecules, including peptide growth factors, pituitary and ovarian hormones, and ECM components, have been implicated [37].

Thrombospondins are now widely considered to be antiangiogenic factors, with inverse relationships reported between proangiogenic factors and TSPs [38, 39]. Although TSPs likely have multiple roles in the regulation of tissue function in the ovary, it is particularly tempting to propose a significant role in the regulation of angiogenesis in the context of the ovarian cycle. In the present study, we show that expression of TSP-1 and CD36 was highest before the development of an extensive vascular network surrounding the follicle. Within the follicle, CD36 expression was also evident in the theca, although not as strongly as observed in the follicle itself. The expression of TSP in small follicles may be one of the factors inhibiting capillary ingrowth into these structures, which normally remain avascular. The findings with TSP-1 are partially consistent with those of Bagavandoss et al. [40], who demonstrated that TSP is expressed in the follicle but observed staining primarily in the basement membrane regions. This discrepancy may result from differences in the timing of observation, because essentially all granulosa cell staining was lost with increasing follicular maturity in the present study. Follicular TSP expression decreased during the period of most extensive vascular growth around the follicle in a time frame that coincides with the expression of proangiogenic factors such as vascular endothelial cell growth factor (VEGF) [41, 42]. However, this association between rapid vascular growth and low levels of TSP is less apparent during the early luteal phase, when a rapid and profound increase in vascular growth occurs. We observed a concurrent increase in the expression of TSP-1 and -2 during this period, when we expected to see low levels of TSP. This temporal pattern of expression is consistent with the findings of others [40], although we did not observe limitation of expression to avascular areas of the corpus luteum. One potential explanation for this apparent paradox of high TSP expression in a period of rapid vascular growth is that a rapid increase in TSP expression may limit overgrowth of the vascular supply in response to high levels of VEGF expression, which occur during this period [43]. Alternatively, TSPs may be multifunctional in the ovary, and expression in the early corpus luteum may play a functional role in the development of luteal cell function, consistent with the expression of CD36 on the surface of luteal cells.

The results of the present study demonstrate that TSP-1 and -2 and their receptor, CD36, are present in the cells and follicular fluid of the rat ovary and that they exhibit distinct patterns of expression. Within the ovaries of rats at various phases of the ovarian cycle, clear regulation of the expression of TSP-1, -2, and CD36 was observed. Both TSP-1 and CD36 were expressed during the early antral stages of follicular development and shortly after formation of the corpus luteum. In addition, both TSP-1 and CD36 appeared to be regulated in a similar fashion, with similar expression of both ligand and receptor on Days 26, 27, and 30. The pattern of protein localization was also largely conserved between TSP-1 and CD36, with expression restricted primarily to the granulosa cells during the early antral phase of the cycle and to the corpus luteum during the early luteal phase. Some staining for TSP-1 and CD36 was also observed in the thecal layers, consistent with the expression on endothelial cells in this highly vascular region. Through interaction with cell-surface receptors, TSP-1 and -2 can stimulate endothelial cell apoptosis [44], suggesting a possible mechanism for TSP-induced inhibition of angiogenesis. Temporal differences in expression were apparent. However, that the cellular distributions of TSP-1 and -2 were similar, with expression localized to the granulosa cells and corpus luteum, is noteworthy. It is possible that the two forms of the protein are involved in mediating different events during the ovarian cycle. Alternatively, the isoforms may be redundant, affecting similar cellular events with variable potencies. Other studies have also demonstrated different expression patterns between TSP-1 and -2, with differences evident throughout murine embryological and postnatal development [45, 46]. As with tissue- and age-specific differences in expression [47], the differences in TSP-1 and -2 expression may be caused by differences in the proximal promoter regions of the two genes.

Our observation that TSP-1 and -2 were differentially regulated during the ovarian cycle led us to investigate the control of TSP expression in vitro in response to gonadotropins. The present study suggests that FSH has no direct effect on TSP-1 expression in vitro and reduces the levels of TSP-2. However, LH treatment of granulosa cells resulted in a marked increase in the expression of both TSP-1 and -2; LH is known to result in the selective transcriptional regulation of a number of genes, including the protease ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin-like motifs) [48]. These studies suggest that both TSPs examined here may be similarly regulated. The decrease observed in TSP-1 expression in cultured granulosa cells in response to FSH was consistent with the in vivo findings. Starting from a relatively high basal level of expression, TSP-1 mRNA levels remained stable during the first 24 h after treatment in a manner virtually identical to the observations in vivo. We hypothesize that secondary factors, initiated by FSH-induced changes in follicular development, may mediate the observed suppression in TSP-1 expression late during follicular development. Secondary factors may include steroid hormones such as estrogens, which regulate TSP-1 in other cell systems, including bone [15] and breast carcinoma [49]. Progesterone may also contribute to the observed regulation of TSP expression, and although progesterone has not previously been shown to modulate TSP-2 expression, it does modulate TSP in the uterus [50], suggesting the potential for TSP regulation by this hormone in the reproductive system.

The studies presented here clearly show that TSP expression in granulosa cells can be modulated by LH in vitro, but the role of the complex and dynamic hormonal milieu of the ovary in the regulation of TSP expression as well as the signaling mechanisms that mediate such changes are clearly subjects for future study. Furthermore, understanding the roles of TSP and its various receptors in the regulation of angiogenesis and ovarian cellular function may provide significant insights regarding the mechanisms that underlie the regular tissue changes that occur throughout the ovarian cycle.


   ACKNOWLEDGMENTS
 
We would like to thank Dr. P. Bornstein (University of Washington) for providing mouse TSP-1 and -2 cDNAs.


   FOOTNOTES
 
1 Financial support for this project was provided by the Lalor Foundation, the Natural Sciences and Engineering Research Council of Canada, and the Institut National de la Santé et de la Recherche Médicale (INSERM). J.L. was the recipient of a Poste-Orange Fellowship from INSERM. Back

2 Correspondence. FAX: 519 767 1450; jlamarre{at}uoguelph.ca Back

Received: 3 May 2002.

First decision: 27 May 2002.

Accepted: 12 June 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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