RAPID COMMUNICATION |
Correspondence to: Nicholas Obermüller, Medical Research Center, Klinikum Mannheim, U. of Heidelberg, 68135 Mannheim, Germany..
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Summary |
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The ovary is one of the organs in which an intrinsic reninangiotensin system (RAS) has been described. Among angiotensin II receptors, the angiotensin II receptor subtype 2 (AT2) is believed to play an important role in mediating or modulating a variety of intraovarian processes. Previous studies were mainly based on ligand binding techniques using AT2 receptor-specific antagonists. Despite the recent cloning of the AT2 gene, no information is available about the exact intraovarian distribution of AT2 mRNA expression. Therefore, we analyzed ovaries from sexually mature, untreated rats by nonradioactive in situ hybridzation using an AT2-specific anti-sense RNA probe. Experiments were performed on perfusion-fixed ovaries obtained from different stages of the estrous cycle. As an important finding, strong AT2 mRNA expression could be demonstrated exclusively in follicular granulosa cells. Follicles containing AT2 mRNA-positve granulosa cells were mainly in the advanced tertiary stage of follicular development, already exhibiting features of atresia. In addition, individual collapsed, definitive atretic follicles also showed strong AT2 mRNA expression solely in granulosa cells. In corpora lutea and in other structures of the ovary, no message for the AT2 receptor could be detected under these conditions. These data may contribute to a better understanding of the effects exerted by an intraovarian RAS. (J Histochem Cytochem 46:865870, 1998)
Key Words: reninangiotensin system (RAS), AT2 receptor, mRNA, in situ hybridization, granulosa cells, atretic follicle, rat ovary
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Introduction |
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Several lines of evidence suggest the existence of an ovarian reninangiotensin system (RAS) including different angiotensin II receptors (for a recent review see
However, the exact sites of AT2 mRNA expression within the adult rat ovary have not yet been determined. In this context, it would be interesting to rule out whether specific AT2 mRNA expression could be confined to characeristic stages of follicular development, including atretic degeneration, or to the formation of corpora lutea. The aim of this study was to identify the cell types that express AT2 mRNA during the estrous cycle in the rat ovary. By using nonradioactive in situ hybridization, this report provides evidence that AT2 mRNA is exclusively expressed in granulosa cells of follicles undergoing atretic degeneration.
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Materials and Methods |
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Animals
Female adult SpragueDawley rats (70100 days) were kept under standard laboratory conditions in the Animal Care Facility in Mannheim. Animals were maintained under controlled conditions of temperature (22C) and lighting (lights on from 06001800 hr) and had free access to rat chow, containing 19% protein, and tapwater. All experiments were conducted in accord with the NIH Guide for the Care and Use of Laboratory Animals. The stage of the estrous cycle was verified daily by examining vaginal smears stained with hematoxylin and eosin. Groups of three animals per stage (proestrus, estrus, metestrus, diestrus) were subjected to subsequent perfusionfixation of the ovaries. Before sacrifice, blood samples were obtained under general anesthesia (see below) from the retro-orbital vein to assess plasma estradiol and progesterone levels. Together with the analysis of the smears and the histological examination of the obtained ovaries, definite assessment of the respective estrous cycle stage was thus achieved.
PerfusionFixation
Rats were deeply anesthetized by IM injection of ketamine (75 mg/kg) and xylazine (6 mg/kg). Animals were perfused retrogradely by cannulation of the distal abdominal aorta just above the beginning of the iliac vessels. Perfusion was conducted at a pressure level of 200220 mmHg with 2% paraformaldehyde in PBS, pH 7.4, for 3 min and subsequently with a 18% sucrose solution dissolved in PBS and adjusted to 800 mOsmol/kg for another 3 min at a pressure of 160 mmHg. After removal, ovaries were divided, mounted quickly on small pieces of styrofoam, and snap-frozen in liquid nitrogen-cooled isopentane. All tissues were stored at -80C until further use.
Preparation of Riboprobes
Full-length rat AT2 cDNA (2.8 KBP) was initially adapted and subcloned into the pCDNAI vector as described in the original report (
Nonradioactive In Situ Hybridization
The in situ hybridization procedure was carried out as described previously (
Subsequently, sections were covered with blocking medium [Buffer I containing 1% blocking reagent (Boehringer Mannheim) and 0.5% bovine serum albumin (BSA)] and incubated for 30 min at RT in a moist chamber. Blocking solution was then drained from the slides and a polyclonal alkaline phosphatase-coupled sheep anti-digoxigenin antibody (Boehringer Mannheim), diluted 1:500 in blocking medium, was applied to the sections. After an incubation of 2 hr at RT the sections were incubated overnight at 4C. The next morning, sections were washed twice for 15 min in Buffer I and equilibrated for 2 min in Buffer II (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl2). For signal development, a color solution [Buffer II containing 0.4 mM nitroblue tetrazolium chloride, NBT (predissolved in 70% dimethylformamide), 0.4 mM 5-bromo-4-chloro-3-indolyl-phosphate, 4-toluidine salt, BCIP or X-phosphate (predissolved in 100% dimethylformamide), and 1 mM levamisole (to inhibit remaining endogenous alkaline phosphatase activity)] was freshly prepared. After covering sections with large amounts of substrate solution, slides were kept at 4C in a moist chamber in the dark. Color reaction was controlled under the microscope and terminated (usually after 1248 hr) by immersing the slides twice for 5 min in Buffer III (100 mM Tris-HCl, 1 mM EDTA, pH 8.0). After rinsing in PBS, sections were mounted in bicarbonate-buffered glycerol, pH 8.6.
For a comprehensive analysis, some sections adjacent to those subjected to the in situ hybridization procedure were stained with hematoxylin and eosin.
Controls
The specifity of the obtained in situ hybridization signal was verified by parallel incubation with anti-sense and sense riboprobes on alternate sections. Throughout all experiments the sense probe did not produce any detectable signal. As further negative controls, some sections were hybridized without anti-sense probe, and others were processed by omission of the anti-DIG antibody. Both controls yielded completely negative results.
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Results |
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An intense in situ hybridization signal for AT2 mRNA could be detected in enlarged antral follicles displaying an advanced stage of follicular development (Figure 1A and Figure 1B). On closer examination, it could be shown that specific labeling of the AT2 RNA probe in a tertiary follicle was exclusively attributable to the granulosa cell layers, whereas theca cells were unreactive for the AT2 RNA probe (Figure 2). However, strong labeling was confined to many but not to all granulosa cells of the respective follicle. Detailed analysis demonstrated that a specific population of follicles contains AT2 mRNA-expressing granulosa cells (Figure 1A, Figure 1B, and Figure 3). Whereas follicles with multiple layers of well-delineated multilaminar cells were devoid of a specific hybridization signal, adjacent tertiary follicles contained AT2 mRNA-expressing granulosa cells. The above-mentioned follicles already showed signs of beginning atresia, including a reduction in the number of granulosa cells, cell shrinking, and nuclear pyknosis in scattered granulosa cells, as shown in Figure 3A and Figure 3B. Moreover, individual collapsed atretic follicles also showed labeling for the AT2 riboprobe in granulosa cells. This is illustrated in an atretic follicle in Figure 3C and Figure 3D, where AT2 mRNA-positve granulosa cells are surrounded by thickened theca cell layers. Extensive hypertrophy of theca cells is a characteristic event during follicle atresia in the rat (
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Discussion |
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This report documents the expression of the AT2 mRNA in ovarian granulosa cells of sexually mature, normally cycling rats, as shown by nonradioactive in situ hybridization. The finding that granulosa cells are responsible for AT2 mRNA expression in the rat ovary is in agreement with previous studies showing AT2 binding sites on rat granulosa cells (
Using morphological criteria for describing the antral follicles that contained AT2 mRNA-positive granulosa cells, it became evident that those follicles exhibited signs of atretic degeneration. Obviously, the granulosa cells that exhibited staining for the AT2 RNA probe in an atretic antral follicle thus classified probably reflected different stages of atretic degeneration. This is likely because AT2 mRNA expression was often seen in a much greater number of granulosa cells in a given follicle than was the number of cells demonstrating typical pycnotic nuclei or apoptotic bodies (for a classification of atresia in the rat see
Our observations, made by high-resolution in situ hybridization analysis clearly localized the AT2 mRNA to rat ovarian granulosa cells of follicles showing the characteristic of atretic degeneration.
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Acknowledgments |
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Supported by the Forschungsfonds der Fakultät für Klinische Medizin Mannheim der Universität Heidelberg.
We thank Sandra Keller for support in DNA preparation.
Received for publication March 23, 1998; accepted March 25, 1998.
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