Immunohistochemical and mRNA Localization of the Angiotensin II Receptor Subtype 2 (AT2) in Follicular Granulosa Cells of the Rat Ovary

Abstract

S U M M A R YA local renin-angiotensin system (RAS), including specific angiotensin II receptor subtypes, is present in the rat ovary. Immunohistochemistry using a polyclonal antibody and mRNA in situ hybridization were performed on perfusion-fixed, paraffinembedded ovaries obtained from untreated sexually mature, normally cycling rats. Immunofluorescent staining revealed an exclusive and distinct labeling of follicular granulosa cells showing a plaque-like expression pattern at the cell borders, being detectable in different stages of atretic degeneration. On adjacent sections the expression of the respective mRNA could be shown in granulosa cells of the same follicle. The AT2 receptor may be implicated in the ovarian atretic process by influencing follicular cell-cell communication. I t is now well established that the ovary possesses an intrinsic renin-angiotensin system (RAS) (for review see Yoshimura 1997). The presence of angiotensin II receptors in the rat ovary has been documented for more than a decade. All these studies have conclusively shown that by far the predominant receptor in the rat ovary is the angiotensin II receptor subtype II (AT2). Studies using binding techniques, autoradiographic analysis, or mRNA in situ hybridization (ISH) experiments have consistently demonstrated that rat granulosa cells of at least atretic follicles express the AT2 receptor (Daud et al. 1988;Tanaka et al. 1995;Obermüller et al. 1998). However, immunohistochemical (IHC) detection of the AT2 receptor in rat granulosa cells has not yet been performed. In particular, its cellular expression site, i.e., cytoplasmic or plasma cell membrane expression, has not been explored. Therefore, we studied the IHC expression of the receptor on rat ovarian sections from normally cycling rats using paraffin-embedded ovarian tissue to obtain high-resolution histomorphology. We have previously shown strong AT2 mRNA expression in rat granulosa cells using ISH on cryostat sections (Obermüller et al. 1998).In the present experiments, ovaries of adult female Sprague-Dawley rats, irrespective of the stage of the estrous cycle, were examined. Female rats were kept under standard laboratory conditions as described earlier (Obermüller et al. 1998). Animals were subjected to perfusion-fixation through the distal abdominal aorta using 2% paraformaldehyde dissolved in PBS, pH 7.4. Ovaries were removed and embedded in paraffin for subsequent procedures. Both ovaries from five animals were investigated. From each ovary, 10-15 sections were cut for analysis.For IHC, sections 2-4-m thick were deparaffinized and washed in bidistilled water. Next, sections were equilibrated in 10 mM citric acid, pH 6.0, for 10 min, microwaved at 500 W for 5 min, and again at 250 W for 10 min. Slides were allowed to cool to room temperature (RT), rinsed in PBS, and incubated

Angiotensin II induces apoptosis in human mural granulosa-lutein cells, but not in cumulus cells

“…These cells secrete and conduct the contents of the ovarian follicle. These include a complete gonadotrophin-regulated plasminogen activator/ plasminogen activator inhibitor system (12), a complete and functional gonadotrophin-sensitive ovarian renin-angiotensin system (OVRAS) (13)(14)(15)(16)(17)(18)(19)(20)(21), and both types of AngII receptors (AT1 and AT2) (21)(22)(23)(24).…”

Expression of angiotensin II type 1 (AT1) and angiotensin II type 2 (AT2) receptors in human granulosa-lutein (GL) cells: correlation with infertility diagnoses

“…Angiotensin II is present in high concentrations in follicular fluid (FF) and granulosa-lutein (GL) cells from patients at the time of egg retrieval (6)(7). Angiotensin II receptors AT1 and AT2 have been identified by RIA and immunohistochemistry in human and other species' ovarian follicular cells (8)(9)(10).…”

O sistema renina-angiotensina na doença periodontal induzida experimentalmente em ratos

“…O protooncogene Mas é sugerido como um receptor funcional de angiotensinas (Jackson et al, 1988 (Campbell, 1987;Phillips, Speakman, Kimura 1993;Obermüller et al, 2004;Leung 2004, Goker et al, 2005Souza et al, 2007;Santos et al 2009). …”