ARTICLE |
Correspondence to: P. Bagavandoss, Dept. of Biological Sciences, Kent State University, Stark Campus, Canton, OH 44720..
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Summary |
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In adult mammals, growth of new vasculature from extant blood vessels (angiogenesis) is rare in the absence of pathology. However, nonpathogenic angiogenesis occurs in the cycling ovary when the avascular postovulatory follicle transforms into a highly vascularized corpus luteum (CL). To improve our understanding of molecular mechanisms that regulate nonpathogenic vascular growth, we characterized the expression of two secreted matricellular proteins associated with angiogenesis, SPARC and thrombospondin (TSP), in postovulatory preluteal follicles and CL of hormone-primed immature rats. By indirect immunofluorescence with specific antibodies, we found SPARC in the cytoplasm of granulosa cells and thecal cells of preluteal follicles, in connective tissue cells of the ovarian interstitium, and in the oocyte nucleus. Administration of a luteinizing stimulus (chorionic gonadotropin) increased the expression of SPARC in granulosa cells. TSP was prominent in the basement membranes of growing follicles. Many cells in the early vascularizing CL expressed both SPARC and TSP. Neovascularization of CL was accompanied by expression of SPARC in nascent vessels and concentration of TSP in central avascular areas. In mature CL, steroidogenic luteal cells expressed both SPARC and TSP. Luteal cells of regressing CL retained SPARC to a variable degree but did not express TSP. The observed changes in expression of SPARC and TSP during development of the CL support distinct roles for these matricellular proteins in nonpathological morphogenesis and angiogenesis. (J Histochem Cytochem 46:10431049, 1998)
Key Words: angiogenesis, ovary, granulosa cells, oocyte, immunofluorescence
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Introduction |
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In adult mammals, the outgrowth of new vasculature from extant blood vessels (angiogenesis) is a characteristic of pathological conditions that include inflammation, wound repair, increases in tumor mass, retinopathies, and hemangiomas. Although nonpathological angiogenesis is rare in most tissues, it occurs in the uterus and ovary during the normal female reproductive cycle. After ovulation, capillaries from the outer thecal layer of the ovarian follicle grow rapidly into the avascular inner layer of granulosa cells. Concomitantly, the granulosa cells proliferate and initiate synthesis of progesterone, an event referred to as luteinization (
The rapid rate of neovascularization of the developing CL and the subsequent regression of vasculature that accompanies degeneration of the CL in the absence of pregnancy make the CL an ideal model for studies of molecular processes that mediate and modulate vascular growth (
Among proteins that influence angiogenesis are the matricellular class of proteins (
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Materials and Methods |
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Animals
LongEvans rats, maintained on a 12-hr:12-hr light:dark cycle at 24C, were fed standard rodent chow (Prolabs; St Louis, MO) and water ad libitum. Immature female rats were obtained as needed by breeding of the adults. Use of animals in this study conformed to the guidelines published by the National Research Council.
Induction of Follicle and CL Development
Immature female rats 25 days of age were made pseudopregnant by injection of 15 IU of pregnant mare's serum gonadotropin (PMSG; Sigma Chemical, St Louis, MO) followed 48 hr later by 5 IU of human chorionic gonadotropin (hCG) (Sigma). Pseudopregnant rats were sacrificed at specific intervals and the ovaries were collected for immunohistochemical analyses. Specific stages of development of follicles or CL were represented by ovaries from a minimum of three animals.
Immunohistochemistry (IHC)
Isolated ovaries were fixed in Bouin's fluid (24 hr at room temperature), dehydrated, embedded in paraffin, and sectioned at 6 µm. Before IHC, deparaffinized sections were incubated for 10 min in 50 mM NH4Cl to quench aldehyde-induced fluorescence. Selected ovaries were frozen in OCT embedding medium (Lab-Tek Products; Naperville, IL) and were sectioned at 8 µm with a cryotome.
Sections processed for IHC were washed in PBS0.2% BSA, blocked with PBS10% BSA (30 min/25C), and exposed to PBS0.2% BSA that contained: (a) a mouse monoclonal antibody (MAb) SSP2 against Domain I of murine SPARC (1:2000 dilution) (
Specificity of TSP and SPARC Antibodies
The specificity of the anti-SPARC and anti-TSP antibodies has been characterized in previous studies. The P12 MAb against TSP immunoprecipitates the 160-kD polypeptide of TSP from human platelet releasates (
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Results |
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Expression of SPARC and TSP During the Growth of Follicles
No fluorescent labeling of sections was observed when secondary antibodies were applied in the absence of primary antibodies (data not shown). In the PMSG-primed ovary, interstitial cells and follicular thecal cells were labeled extensively with anti-SPARC IgG (Figure 1A). Follicular granulosa cells expressed SPARC to a lesser degree than was observed in thecal cells or interstitial cells (Figure 1A). After injection of hCG, expression of SPARC by follicular granulosa cells increased relative to that of thecal cells (Figure 1B). Granulosa cells of the corona radiata that surrounded maturing oocytes within antral follicles (Figure 1C) exhibited significant levels of SPARC in the cytoplasm. SPARC was not found in the cytoplasm of oocytes. However, the nuclei of oocytes were strongly immunopositive for SPARC (Figure 1C). In contrast, granulosa cells did not express SPARC in their nuclei. TSP was expressed at high levels in the basement membranes of specific growing follicles (Figure 2). However, this expression was variable (not shown).
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SPARC and TSP in the Developing CL
During the earliest stages of development of the CL, granulosa cells did not express detectable levels of SPARC (not shown). Granulosa cells in CL undergoing neovascularization produced both SPARC and TSP, and many cells expressed both proteins concomitantly (Figure 3). With the appearance of well-defined vasculature in CL, SPARC was expressed by microvessels throughout the CL as well as in the avascular center of the CL (Figure 4A). TSP was concentrated only in the avascular center of the CL (Figure 4B).
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SPARC and TSP in Mature and Regressing CL
In mature CL, expression of SPARC by vasculature was significantly diminished. SPARC and TSP were both expressed in the cytoplasm of luteal cells (Figure 5A and Figure 5C). In rats, regression of the CL is maximal by Day 13 of PMSGhCG-induced pseudopregnancy because serum progesterone returns to basal levels at this time (
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Discussion |
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We have examined the expression of the matricellular proteins SPARC and TSP in the developing follicle and CL of the rat. The polyclonal anti-TSP antibodies used in this study do not distinguish between TSP-1 and -2. However, they are likely to recognize TSP-1 preferentially, based on confirmation by in situ hybridization studies (
The variable expression of TSP by follicles at different stages of development is indicative of hormonal regulation. Indeed, expression of TSP by isolated rat granulosa cells in vitro is modulated by follicle-stimulating hormone (
SPARC is expressed in vivo where cells are proliferating, migrating, and undergoing structural reorganization, e.g., during embryonic development or during repair of wounds (
We find that the nuclei of maturing oocytes are markedly positive for SPARC. SPARC has also been found in nuclei of embryonic chick mesenchymal cells (M. Gooden, R. Vernon, and H. Sage, unpublished observations). Because extracellular SPARC is taken up by embryonic chick cells in vitro and is subsequently transported to the nucleus (M. Gooden, R. Vernon, and H. Sage, unpublished observations), SPARC within the oocyte nucleus might derive from extracellular sources, such as adjacent granulosa cells that provide trophic support to the oocyte. The function of SPARC in the oocyte nucleus is unknown. However, it is noteworthy that the protein calmodulin, which contains Ca+2 binding loops similar to those of SPARC, is implicated in the resumption of meiosis of the starfish oocyte (
In developing CL, neovascularization of the granulosa layer progresses centripetally from the parent vasculature of the theca, which results in a steady reduction in the size of the central avascular area. In early stages of vascularization of the CL, we found TSP and SPARC in most preluteal granulosa cells. In contrast, in late CL where neovascularization was nearly complete, TSP was confined to the central avascular area, a result consistent with an anti-angiogenic function for TSP. TSP causes endothelial cells to decrease expression of uPA and to increase expression of PAI-1, responses that result in lower levels of matrix proteolysis and diminished angiogenesis (
Previous studies have reported that SPARC is expresssed by endothelial cells undergoing angiogenic morphogenesis in vitro and in vivo (
TSP was expressed by luteal cells of mature, functional CL but was lacking in luteal cells within regressive CL. Luteal cells in vivo and in vitro synthesize tissue-type plasminogen activator (tPA), which inhibits the production of progesterone by luteal cells (
A recent study has reported the expression of SPARC by small luteal cells of the ovine CL (
In summary, we have shown that the developing follicle and CL of the rat ovary exhibit distinctive staining patterns for two significant members of the matricellular class of extracellular proteins, i.e., SPARC and TSP. Although there was near-coincident expression of these proteins in granulosa and steriodogenic luteal cells, the distribution of SPARC and TSP in the invading vasculature of the CL was unique and moreover, was consonant with their known functions in angiogenesis. Therefore, preluteal follicles and developing CL provide a singular example of nonembryonic, nonpathologic angiogenesis in which two matricellular proteins might exert opposing effects by virtue of their distinctive localizations.
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Acknowledgments |
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We are indebted to Dr B.C. Bruot for providing his laboratory facilities for conducting this research and to Dr Jennifer Marcinkiewicz for graciously sharing her histology facility. We also thank Drs V.M. Dixit and W.A. Frazier for providing the rabbit polyclonal antibody to TSP.
Received for publication January 30, 1998; accepted May 7, 1998.
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