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Endocrinology, doi:10.1210/en.2007-0835
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Endocrinology Vol. 149, No. 1 185-192
Copyright © 2008 by The Endocrine Society

Thrombospondin-1 Expression Is Increased during Follicular Atresia in the Primate Ovary

Fiona H. Thomas, Helen Wilson, Audrey Silvestri and Hamish M. Fraser

Medical Research Council Human Reproductive Sciences Unit, University of Edinburgh Centre for Reproductive Biology, The Queen’s Medical Research Institute, Edinburgh EH16 4TJ, United Kingdom

Address all correspondence and requests for reprints to: Fiona H. Thomas, Medical Research Council Human Reproductive Sciences Unit, University of Edinburgh Centre for Reproductive Biology, The Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom. E-mail: f.thomas{at}hrsu.mrc.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thrombospondin (TSP)-1 is an antiangiogenic extracellular matrix glycoprotein that modulates several aspects of cellular function. The aim of this study was to determine the pattern of TSP-1 mRNA and protein expression as well as expression of its receptor CD36 in the marmoset ovary and to investigate the effects of inhibition of gonadotropins or VEGF activity on TSP-1 and CD36 expression in vivo. GnRH antagonist or VEGF Trap, a soluble decoy receptor, was administered on d 0 of the follicular phase of the cycle, and ovaries were collected at the end of the follicular phase (d 10). TSP-1 mRNA and protein were present in granulosa cells of preantral and antral follicles, with the highest staining at the late secondary and tertiary stages. Moreover, expression of TSP-1 mRNA and protein was significantly increased in tertiary follicles undergoing atresia. CD36 protein was detected in granulosa cells of preantral and antral follicles as well as in endothelial cells of large vessels. Inhibition of gonadotropin secretion or VEGF activity had no effect on TSP-1 expression; however, expression of CD36 protein was inhibited by the VEGF Trap. In conclusion, TSP-1 may be involved in the cessation of angiogenesis in follicles undergoing atresia; alternatively, TSP-1 may act on granulosa and/or endothelial cells to promote follicular atresia in the ovary. Angiogenesis is likely to involve a balance between pro- and antiangiogenic factors. Our results suggest that loss of VEGF activity does not regulate TSP-1 expression directly but may influence TSP-1 activity via down-regulation of the CD36 receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OVARIAN FOLLICULOGENESIS is regulated by both endocrine and intraovarian mechanisms that coordinate the processes of cellular proliferation and differentiation (1). This regulation occurs at various levels, with a shifting contribution by endocrine, autocrine, and paracrine factors. The availability of an adequate vascular supply is necessary to provide endocrine and intraovarian signals during follicular development (2, 3). In the adult, the ovary is distinctive in that it is a tissue that undergoes physiological angiogenesis, in which blood vessels are programmed to develop and regress in a cyclic manner. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that stimulates endothelial cell proliferation and migration and increases vascular permeability (4, 5, 6). Through the use of specific antagonists administered at selected stages of the ovulatory cycle in marmosets, we have established a role for VEGF in the regulation of cyclical angiogenesis that takes place in the developing follicle and corpus luteum (2, 4). By inhibiting VEGF using the VEGF Trap, these studies have established a requirement for VEGF during follicular vascularization, antrum formation, and ovulation (2, 4). In addition to these observations, proliferation of theca, endothelial, and granulosa cells is inhibited by the VEGF Trap (4). We have also previously shown that treatment of marmosets with the VEGF Trap or a GnRH antagonist for the entire follicular phase (d 0–10 of the cycle) results in the absence of dominant preovulatory follicles, whereas in the remaining antral follicles, granulosa, theca, and endothelial cell proliferation is reduced (7).

Because physiological angiogenesis in the ovary is tightly controlled, it is reasonable to assume that antiangiogenic factors are involved in this process and that there may be cross-talk between pro- and antiangiogenic factors. Several naturally occurring inhibitors of angiogenesis have been identified in mammalian tissues and are thought to maintain the quiescence of the normal vasculature (8). Among these are members of the thrombospondin (TSP) family, TSP-1 and TSP-2. These are large glycoproteins secreted by several cell types and found in the extracellular matrix (9). The effects of TSPs are mediated through interaction with the cell surface receptors CD36 and integrin-associated protein (IAP, also known as CD47) (10, 11, 12). TSP-1 suppresses angiogenic responses in animal models (13), and both in vivo and in vitro studies have shown that treatment with TSP-1 renders endothelial cells unable to respond to a wide variety of inducers of angiogenesis (14). TSP-1 has also been reported to induce apoptosis in cultured cells derived from the vascular endothelium (15), suggesting that this factor may inhibit angiogenesis by destroying the microvascular endothelial cells that are forming new vessels (16).

TSP localization has been previously investigated in the rat (17) and cow ovary (18), with the pattern of expression being inversely correlated to the expression of VEGF (18). In addition, a role for TSP-1 in inhibition of VEGF levels has been demonstrated in TSP-1 knockout mice, which exhibit follicular hypervascularization and reduced litter size (19). Thus, TSPs may be involved in suppressing the development of blood vessels during the early stages of follicular development.

The regulation of TSP expression in reproductive tissues appears to vary according to species and/or experimental model. In the human endometrium, progesterone has been shown to stimulate TSP mRNA synthesis (20). In rat immortalized granulosa cells in vitro, the expression of both TSP-1 and -2 mRNA is increased in response to LH stimulation; however, TSP-2 is suppressed by FSH (17). In cultured bovine granulosa cells, TSP-1 and -2 protein production is stimulated by FSH, whereas LH has no effect (18). Thus, more information on the regulation of TSP-1 mRNA and protein is required, with the marmoset ovary providing a novel in vivo model for studying these mechanisms.

TSP-1 mRNA and protein localization in the ovary has not been previously described in primates. The present study therefore aims to determine the pattern of expression of TSP-1 and its receptor CD36 during normal follicular development in the marmoset ovary. In addition, using the established VEGF Trap and GnRH antagonist models, we will determine whether the expression of TSP-1 and CD36 are influenced by inhibition of VEGF activity or loss of gonadotropin stimulation in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult female common marmoset monkeys (Callithrix jacchus), 2–3 yr old with a body weight of approximately 350 g and regular (28-d) ovulatory cycles as determined by plasma progesterone concentrations of blood samples collected three times per week (21), were housed together with a younger sister or prepubertal female as described previously (22).

Treatments
Experiments were carried out in accordance with the Animals (Scientific Procedures) Act, 1986, and were approved by the Local Ethical Review Process Committee. To synchronize follicular recruitment, selection, and ovulation during the treatment cycle, and to render the length of the luteal phase similar to that of higher primates, marmosets were treated with 1 µg prostaglandin F2{alpha} analog (cloprostenol, Planate; Coopers Animal Health Ltd., Crewe, UK), im on d 13–15 of the 20-d luteal phase to induce luteolysis (23, 24). The day of prostaglandin injection was designated follicular d 0. This is associated with follicular recruitment, followed by selection on d 5 and ovulation around d 10 (24).

To block the action of VEGF, we employed the VEGF Trap, a soluble decoy receptor created by fusing the extracellular domains of the human VEGF receptors (VEGFR1 and -2) to the Fc portion of a human Ig. The incorporation of the Fc domain results in homodimerization of the recombinant protein, creating a high-affinity VEGF Trap (25). The VEGF trap binds all isoforms of VEGF-A, VEGF-B, and placental growth factor. The VEGF Trap was administered as a single sc injection at 25 mg/kg on d 0 (n = 4) of the follicular phase as described previously (26, 27), and ovaries were collected on d 10, corresponding to the periovulatory period in control animals.

To suppress FSH and LH secretion from the pituitary (28, 29, 30), a GnRH antagonist, antarelix (31), was used as described previously (7, 27). Antarelix was injected at a dose of 12 mg/kg sc on follicular d 0 (n = 4). As for the VEGF Trap group, ovaries were collected on d 10 of the follicular phase. Ovaries from control marmosets (n = 4) were collected during the late follicular phase of the cycle.

At the end of the treatment periods, animals were sedated using 100 µl ketamine hydrochloride (Parke-Davis Veterinary, Pontypool, UK) and euthanized with an iv injection of 400 µl Euthetal (sodium pentobarbitone; Rhone Merieux, Harlow, UK). After cardiac exsanguination, ovaries were removed, weighed, and immediately fixed in 4% neutral buffered formalin. After 24 h, the ovaries were transferred to 70% ethanol, dehydrated, and embedded in paraffin according to standard procedures.

Immunohistochemistry
Ovaries were embedded and serially sectioned, and tissue sections (5 µm) were placed onto BDH SuperFrost slides (Merck Co., Inc., Poole, UK). After dewaxing in xylene and rehydration in a series of ethanols, expression of TSP-1 or CD36 protein in the control and treated marmoset ovaries was detected by immunohistochemistry using the Bond automated immunohistochemistry system (Vision BioSystems, Newcastle upon Tyne, UK). For TSP-1, after dewaxing in xylene and rehydration in ethanol, antigen retrieval was performed by pressure cooking (Tefal pressure cooker; Tefal, Essex, UK) in 0.01 M citrate buffer (pH 6) for 6 min at high pressure. Sections were left for 20 min in hot buffer and washed in Tris-buffered saline (0.05 mol/liter Tris and 9 g/liter NaCl), before being subjected to automated immunohistochemistry using the Polymer Refine kit (Vision BioSystems). The primary antibody to human TSP-1 (mouse monoclonal antibody; Abcam, Cambridge, UK) was diluted 1:50 in Bond antibody diluent and incubation was carried out for 2 h at room temperature. For the negative controls, primary antibody was omitted. CD36 immunostaining was carried out as for TSP-1. The primary antibody to CD36 (mouse antimouse CD36 monoclonal antibody; BD Biosciences, Oxford, UK) was diluted 1:50 in Bond antibody diluent, and incubation was carried out for 30 min at room temperature. For the negative controls, primary antibody was omitted.

To assess follicular atresia, immunohistochemistry for activated caspase-3 (Asp175; New England Biolabs, Hitchin, UK) was performed as described previously (26, 27).

Quantification of immunohistochemistry
Right and left ovaries from control animals (n = 4), VEGF Trap-treated animals (n = 4), and GnRH antagonist-treated animals (n = 4) were examined for TSP-1 and CD36 expression. Stages of follicular development were defined as previously reported (4, 21, 27), i.e. primary (oocyte surrounded by one granulosa cell layer), early secondary (two to four granulosa cell layers, no antrum), late secondary (more than four granulosa cell layers, no antrum), and tertiary (follicles containing an antrum < 2 mm). Only those follicles with a visible oocyte containing a nucleus were considered to ensure proper follicular classification.

TSP-1 expression was investigated in early secondary, late secondary, and tertiary follicles of marmoset control ovaries (n = 4 animals) and ovaries treated with VEGF Trap (n = 4 animals) or GnRH antagonist (n = 4 animals), with the intensity of TSP-1 expression being classified in each follicle using a visual scoring system (1 = weak; 2 = moderate; 3 = strong staining). The percentage of follicles at each intensity was then calculated for each follicle stage and expressed as a mean HSCORE value (32). HSCORE is a semiquantitative analysis that has been shown to have low intra- and interobserver error (33). In the initial analysis, follicles were classed as atretic if they had more than 5% pyknotic nuclei. This classification is based on previously described criteria for the measurement of follicular cell death (34). Follicles with morphological signs such as granulosa cell shrinkage and detachment of cells from the oocyte or basement membrane were also classed as atretic. Follicles were analyzed in three representative sections per ovary, and two ovaries per animal were used. The numbers of follicles analyzed were as follows: controls (late secondary n = 59; tertiary n = 9), VEGF Trap (late secondary n = 57; tertiary n = 5), and GnRH antagonist (late secondary n = 36; tertiary n = 18). The extent of follicular atresia and the relationship between cell death and TSP-1 expression was further investigated by analysis of activated caspase-3 expression in tertiary follicles within the ovaries of five animals selected at random (three sections per ovary). The ovary sections chosen were adjacent to the sections analyzed for TSP-1 mRNA expression so that accurate comparisons could be made.

CD36 expression was assessed in control ovaries (n = 4 animals) and ovaries treated with VEGF Trap (n = 3 animals) or GnRH antagonist (n = 3 animals). Initially, the presence or absence of staining was noted in granulosa cells, endothelial cells of vessels within the ovarian stroma, surface epithelium, stroma, and thecal layer. Subsequently, the number of tertiary follicles expressing CD36 in the granulosa cell layer was counted and expressed as a percentage of the total number of tertiary follicles in each treatment group. The numbers of tertiary follicles analyzed were as follows: controls (n = 11), VEGF Trap (n = 14), and GnRH antagonist (n = 9).

In situ hybridization
The cDNA fragment used as a template for in situ hybridization was a 1.3-kb EcoR1-EcoR1 fragment of human TSP-1 (35) cloned into the vector pGEM-2 in an antisense orientation relative to SP6. Detection of expression of TSP-1 mRNA was performed as described previously (36) with some modifications. Sense and antisense probes were prepared using an RNA transcription kit (Ambion Inc., Austin, TX) and were labeled with [35S]uridine 5'-triphosphate (Perkin-Elmer, Beaconsfield, UK). Deparaffinized sections were treated with 0.1 M HCl and then digested in proteinase K (5 µg/ml; Sigma, Poole, UK) for 30 min at 37 C followed by 5 min in triethanolamine buffer and 10 min in triethanolamine/acetic anhydride. After prehybridization for 2 h at 55 C, hybridization was performed in a moist chamber overnight at 55 C. High-stringency posthybridization washes and ribonuclease A treatment were carried out to remove excess probe. Slides were then dehydrated, dried, and dipped in Ilford G5 liquid emulsion (Agar Scientific, Stansted, UK). Slides were developed after 7 wk (Kodak D19 developer; Calumet Photographic Ltd., Milton Keynes, UK) and fixed (Kodak GBS; Sigma). Slides were counterstained with hematoxylin (BIOS Europe Ltd., Lancashire, UK), dehydrated, and mounted.

Expression of TSP-1 mRNA in late secondary and tertiary follicles was analyzed qualitatively under light-field and quantitatively under dark-field conditions. Adjacent sequential sections were used for the immunohistochemical and in situ hybridization analyses. Grain density (number of grains per square micrometer) was calculated using an image analysis system linked to an Insight camera (Diagnostic Instruments, Inc., Sterling Heights, MI), and the data were processed using Image-Pro software (Media Cybernetics, Bethesda, MD).

Statistical analyses
The percentage of tertiary follicles showing signs of follicular atresia was compared between treatment groups using {chi}2 analysis followed by Fisher’s exact test. For quantification of TSP-1 mRNA and protein expression, mean values (grain density or HSCORE) for follicles at each stage of development were compared between groups using one-way ANOVA, followed by Tukey’s multiple comparison test (GraphPad Prism). To determine whether TSP-1 expression was associated with increased cell death, a correlation was performed between the mean percent activated caspase-3 expression and mean TSP-1 mRNA expression (Microsoft Excel), and this correlation was tested for significance taking into account the sample size and correlation coefficient. For quantification of the proportion of ovaries or tertiary follicles expressing CD36, {chi}2 analysis was carried out followed by Fisher’s exact test to determine differences between treatment groups. P values < 0.05 were accepted as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of TSP-1 mRNA and protein in marmoset ovaries
Low levels of TSP-1 mRNA expression were observed in granulosa cells of healthy secondary and tertiary follicles within the marmoset ovary (Fig. 1Go, A and B). TSP-1 mRNA expression peaked in granulosa cells at the tertiary stage, with lower expression as follicular development progressed to the preovulatory stage (Fig. 1Go, A–C). TSP-1 mRNA expression was highest in granulosa cells of tertiary follicles undergoing atresia (Fig. 1DGo). A similar pattern of TSP-1 protein expression was observed (Fig. 2Go).


Figure 1
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  		FIG. 1. Representative sections showing TSP-1 mRNA expression in granulosa cells (arrows) of secondary (A and E), tertiary (B and F), preovulatory (C and G), and atretic (D and H) follicles under light-field (A–D) and dark-field (E–H) illumination. In healthy follicles, expression peaked at the tertiary stage (B and F), with reduced expression at the preovulatory stage (C and G). However, the highest expression was observed in atretic follicles (D and H). Bar, 80 µm.

 

Figure 2
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  		FIG. 2. Representative sections showing expression of TSP-1 protein in granulosa cells of follicles in the whole ovary (A) (arrows) and in secondary (B), tertiary (C), preovulatory (D), and atretic follicles (E). In healthy follicles, expression peaked at the tertiary stage (C) and decreased in preovulatory follicles (D). The highest expression was observed in atretic follicles (E). Note that staining in atretic follicles is also evident at the top right corner of B and C. Negative controls for each stage are shown (F–I). Bars, 700 µm (A), 80 µm (B–E, G, and I), and 100 µm (F and H).

 
Expression of CD36 protein in marmoset ovaries
CD36 protein was expressed in granulosa cells, endothelial cells of vessels within the ovarian stroma, surface epithelium, and the stroma of control ovaries (Fig. 3Go and Table 1Go). A small proportion of control ovaries also had CD36 expression in the thecal layer (Table 1Go).


Figure 3
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  		FIG. 3. A–C, Representative sections showing CD36 protein expression in the whole ovary (A) and in granulosa cells (arrows) of tertiary follicles from control animals (B) and animals treated with VEGF Trap (C) or GnRH antagonist (D); E, negative control follicle; F, expression of CD36 protein in the ovarian vasculature. Bars, 400 µm (A), 200 µm (B–D and F), and100 µm (E).

 

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  		TABLE 1. Percentage of ovaries with CD36 expression in granulosa, theca, vasculature, stroma, and surface epithelium of the ovary.

 
Effect of treatment with VEGF Trap and GnRH antagonist on TSP-1 and CD36 expression
There was no significant effect of treatment with VEGF Trap or GnRH antagonist on the expression of TSP-1 mRNA or protein in follicles at the late secondary or tertiary stage (results not shown). In addition, there was no significant effect of treatment on the percentage of tertiary follicles showing signs of follicular atresia (Fig. 4AGo). However, of the tertiary follicles undergoing atresia, all had either moderate or high TSP-1 protein expression in their granulosa cells (Fig. 4BGo), an effect that was independent of treatment. Further analysis was carried out to determine the stage of follicle atresia that TSP-1 expression was increased. Comparison of mean TSP-1 mRNA expression and mean activated caspase-3 protein expression in adjacent ovarian sections from five animals revealed that there was a strong correlation between cell death and TSP-1 mRNA expression (P < 0.05; Fig. 4CGo).


Figure 4
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  		FIG. 4. A, Quantification of the percentage of tertiary follicles undergoing atresia in control ovaries and ovaries treated with VEGF Trap or GnRH antagonist (GnRH antag); B, quantification of the intensity of TSP-1 protein expression in follicles undergoing atresia (different letters indicate significant differences at P < 0.05); C, comparison of mean TSP-1 mRNA expression and mean activated caspase-3 protein expression in tertiary follicles from five animals reveals a strong correlation between TSP-1 mRNA expression and cell death (r = 0.92; P < 0.05).

 
Lastly, treatment with VEGF Trap resulted in the loss of expression of CD36 in all cell types within the ovary (Table 1Go), but administration of GnRH antagonist had no significant effect compared with controls. When CD36 expression in the granulosa cells of tertiary follicles was quantified for each treatment group, there was significantly higher expression in controls and GnRH antagonist-treated ovaries, compared with VEGF Trap-treated ovaries (P < 0.05; Table 2Go).


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  		TABLE 2. Percentage of tertiary follicles expressing CD36 in granulosa cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TSP-1 was localized to granulosa cells of secondary and tertiary follicles within the marmoset ovary, with decreased expression in follicles at the preovulatory stage. This pattern of expression is in agreement with the expression pattern seen in the bovine ovary (18). The TSP receptor CD36 was also expressed in granulosa cells as well as in other cell types within the marmoset ovary, including endothelial cells. In addition, we have shown for the first time that TSP-1 mRNA and protein are up-regulated in follicles undergoing atresia, with a positive correlation between TSP-1 expression and increasing follicular atresia.

Previous studies have investigated expression of TSP-1 in rodent and bovine ovaries (17, 18). In the rat, TSP-1 and CD36 are expressed mainly in the granulosa cells of preantral and early antral follicles, with TSP-2 being expressed in the early corpus luteum (17). In that study, it was noted that there was no apparent difference in TSP-1 or -2 or CD36 immunostaining between healthy and atretic follicles, although this was not quantified (17). In the cow ovary, TSP and CD36 protein were colocalized in the granulosa cells of small and medium antral follicles (0.5–1 cm) (18), with reduced expression of TSP mRNA and protein in larger follicles. We have observed a similar pattern of expression in the marmoset ovary. In addition, we have shown that TSP-1 mRNA and protein are up-regulated during follicular atresia.

Because atretic follicles had increased TSP-1 mRNA and protein expression in the present study, TSP-1 may be involved in the cessation of angiogenesis in follicles undergoing atresia. Alternatively, TSP-1 may be an autocrine/paracrine factor acting on granulosa and/or endothelial cells to promote follicular atresia in the ovary, because CD36 was found to be present in both granulosa and endothelial cells. Analysis of the antiangiogenic mechanisms of TSP-1 in human dermal microvascular endothelial cells has shown that induction of apoptosis by TSP-1 required the sequential activation of the CD36 receptor, p59fyn, caspase-like proteases, and p38 MAPKs (16). In addition, in cultures of spontaneously immortalized rat granulosa cells, TSP-1 decreased expression of the antiapoptotic factor bcl-2 and increased levels of Fas, a proapoptotic factor (19), suggesting that TSP-1 may regulate the process of apoptosis in the ovary. VEGF has been implicated as a cytoprotective factor in the bovine ovary via VEGFR2 (Flk-1/KDR) expressed in granulosa cells (37). Because TSP-1 has been shown to inhibit VEGF levels in the mouse ovary (19), this mechanism may be relevant to the proposed role of TSP-1 in follicle atresia in the present study. However, because VEGFR2 has not been shown to be expressed in granulosa cells in the marmoset ovary, this hypothesis requires further investigation. Elevated VEGF levels are associated with polycystic ovary syndrome (38), which is characterized by an increased number of small antral follicles and reduced follicular atresia (39). The in situ expression pattern of TSP-1 mRNA and protein in polycystic ovary syndrome has yet to be determined, but down-regulation of TSP-1 expression/activity in these ovaries is an interesting possibility, requiring further investigation.

Because angiogenesis must be tightly controlled, it is reasonable to assume that both pro- and antiangiogenic factors are involved in this process and that there may be cross-talk between VEGF and TSP-1. TSP-1 modulates endothelial cell invasion and morphogenesis in vitro by up-regulation of matrix metalloproteinase-9 (40). VEGF has also been reported to regulate matrix metalloproteinase-9 expression (41), suggesting that TSP and VEGF can act on similar pathways to influence angiogenesis. Additional evidence for interaction between VEGF and TSP has been provided in a recent study, which has shown that TSP-1 can inhibit VEGF levels directly in the ovary by binding and internalization of VEGF (19). We have now investigated for the first time the possibility that inhibition of VEGF influences TSP-1 expression in the marmoset ovary. Our findings show that although treatment with the VEGF Trap does not regulate TSP-1 mRNA or protein expression directly, it does result in inhibition of expression of the CD36 receptor. Recently, activation of CD36 by TSP-1 has been reported to down-regulate VEGFR2 in endothelial cells in vitro (42). Taken together, these findings suggest that a feedback loop may exist between VEGF and TSP, with VEGF stimulating TSP activity via up-regulation of CD36, which in turn inhibits VEGF activity. Such a mechanism may be critical in the ovary where angiogenesis is tightly controlled throughout the cycle.

The role of gonadotropins in the regulation of TSP-1 expression has been investigated previously in cultured granulosa cells from rodent and bovine ovaries, with differing results (17, 18). In the rat, LH appears to play a major role in promoting expression of TSP-1 and -2 (17), whereas in bovine granulosa cells, FSH stimulates TSP expression but LH has no effect (18). These differences have been attributed to species differences in the timing of expression of LH receptors during the rodent and bovine reproductive cycles (18). It has also been suggested that FSH-induced changes in follicular development, such as production of estrogens and progesterone, may mediate suppression of TSP-1 expression during late follicular development (17). In other organs such as breast, estradiol has been reported to up-regulate VEGF (43) and repress TSP-1 expression (44). Using a GnRH antagonist, we have shown that withdrawal of gonadotropins does not play a major role in the regulation of TSP-1 and CD36 expression in vivo in the marmoset ovary. This corresponds with the finding that TSP-1 is expressed at the preantral and early antral stage of development, before gonadotropin dependence has been established. However, it should be noted that the GnRH antagonist treatment also results in suppression of estradiol and progesterone production by the ovary (7). Given the differential roles of gonadotropins and steroid hormones on regulation of TSP-1 expression (17, 18, 44), regulation of TSP-1 expression is likely to involve a combination of stimulatory and inhibitory factors, and more research is required to delineate the mechanisms involved.

In summary, we have described the pattern of TSP-1 mRNA and protein expression during follicular development in the marmoset ovary and have demonstrated an up-regulation of TSP-1 expression during progression of follicular atresia. TSP-1 may be involved in the cessation of angiogenesis in follicles undergoing atresia; alternatively, TSP-1 may be an autocrine/paracrine factor acting on granulosa and/or endothelial cells to promote follicular atresia in the ovary. Angiogenesis is likely to involve a balance between pro- and antiangiogenic factors. Our results suggest that although loss of VEGF activity does not regulate TSP-1 expression directly, it may influence TSP-1 activity via down-regulation of the CD36 receptor.


   Acknowledgments
 
We thank Dr. J. S. Rudge, Dr. S. J. Weigand, and Regeneron Pharmaceuticals Inc. (Tarrytown, NY) for expert advice and gift of the VEGF Trap and Dr. J. Lawler (Harvard University) and Dr. A. M. Schor (University of Dundee) for the gift of the TSP-1 in situ probe. We also thank Keith Morris and staff for animal care, Nancy Evans for help with TSP-1 immunocytochemistry, and Ian Swanston for the progesterone assays.


   Footnotes
 
Disclosure Statement: F.T., H.W., A.S., and H.F. have nothing to declare.

First Published Online September 20, 2007

Abbreviations: TSP, Thrombospondin; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.

Received June 22, 2007.

Accepted for publication September 12, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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