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. 2009 Jul;57(7):633-41.
doi: 10.1369/jhc.2009.953059. Epub 2009 Mar 2.

Immunohistochemical demonstration of the mineralocorticoid receptor, 11beta-hydroxysteroid dehydrogenase-1 and -2, and hexose-6-phosphate dehydrogenase in rat ovary

Elise P Gomez-Sanchez  1 , Miriam T Gomez-Sanchez, Angela F de Rodriguez, Damian G Romero, Mary P Warden, Maria W Plonczynski, Celso E Gomez-Sanchez
Affiliations

Immunohistochemical demonstration of the mineralocorticoid receptor, 11beta-hydroxysteroid dehydrogenase-1 and -2, and hexose-6-phosphate dehydrogenase in rat ovary

Elise P Gomez-Sanchez et al. J Histochem Cytochem. 2009 Jul.

Abstract

An IHC survey using several monoclonal antibodies against different portions of the rat mineralocorticoid receptor (MR) molecule demonstrated significant specific MR immunoreactivity in the ovary, prompting further study of the localization of MR and of determinants of extrinsic MR ligand specificity, 11beta-hydroxysteroid dehydrogenase (11beta-HSD) types 1 and 2, and hexose-6-phosphate dehydrogenase (H6PDH). MR expression (real-time RT-PCR and Western blot) did not differ significantly in whole rat ovaries at early diestrus, late diestrus, estrus, and a few hours after ovulation. MR immunostaining was most intense in corporal lutea cells, light to moderate in oocytes and granulosa cells, and least intense in theca cells. Light immunoreactivity for 11beta-HSD2 occurred in most cells, with some mural granulosa cells of mature follicles staining more strongly. The distribution of immunoreactivity for 11beta-HSD1 and H6PDH required to generate NADPH, the cofactor required for reductase activity of 11beta-HSD1, was similar, with the most-intense staining in the cytoplasm of corporal lutea and theca cells and light or no staining in the granulosa and oocytes. MR function in the ovary is as yet unclear, but distinct patterns of distribution of 11beta-HSD1 and -2 and H6PDH suggest that the ligand for MR activation in different cells of the ovary may be differentially regulated.

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Figures

Figure 1
Figure 1
Densitometric summary, expressed as the ratio of mRNA for mineralocorticoid receptor (MR) (top), 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) (middle), and 11β-HSD2 (bottom) to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat ovaries (on days 1–4 of the estrus cycle), super-ovulated ovaries, colon, and kidney. Estrus cycles of the rats were synchronized with luteinizing hormone-releasing hormone (LHRH), and ovaries were taken at day 1 (early diestrus), day 2 (late diestrus), day 3 (estrus), and day 4 (late estrus). Sup-Ov: super-ovulated ovaries taken from rats 8–12 hr after coitus after super-ovulation induced by the administration of LHRH (40 μg/rat; ∼200 μg/kg on day 4), pregnant mare serum gonadotropin (300 IU/kg on day 2), and human chorionic gonadotropin (300 IU/kg on day 0). Col: colon; Kid: kidney. Error bars represent standard error of the mean. *Denotes significance compared with the values of mRNA from super-ovulated ovaries.
Figure 2
Figure 2
Western blot analysis for MR in hippocampus and ovary (A), and for 11β-HSD2 (B), 11-βHSD1 (C), and H6PDH (D) in kidney and ovary. Whole-tissue protein was used for gel A, microsomal protein was used for gels B–D.
Figure 3
Figure 3
Photomicrographs of immunohistochemical detection of the MR in a rat ovary using two different monoclonal antibodies. (A) Control, no primary antibody. (B–F) Antibody against aa 79–97 of the MR sequence. (G–K) Antibody against aa 64–82. (C,D) Higher magnifications of the boxed areas in B and C, respectively. (F) Higher magnification of the boxed area in E. (J,K) Successive magnifications of I and J. A, follicular antrum; CL, corpora lutea; T, theca cells; G, granulosa cells; M, macrophage.
Figure 4
Figure 4
Photomicrographs of immunohistochemical detection of 11β-HSD1, 11β-HSD2, and H6PDH in a rat ovary. (A,E,I) Negative controls for 11β-HSD1, 11β-HSD2, and H6PDH, respectively. (A–D) 11β-HSD2. (C,D) Higher magnifications of areas boxed in B. The follicle in upper left of B appears to be atretic and includes several active macrophages. (E–H) 11β-HSD1. (G) Higher magnification of boxed area in F. (H) Higher magnification of a corpora lutea showing intense 11β-HSD1 immunoreactivity in a few cells. (I–M) H6PDH. (K,M) Higher magnifications of boxed areas in J and L, respectively, showing the oocyte with preovulatory cumulus cells (K) and corpora lutea (M). A, follicular antrum; CL, corpora lutea; T, theca cells; G, granulosa cells; M, macrophage.
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