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. 2013 Jun;154(6):2174-87.
doi: 10.1210/en.2012-2256. Epub 2013 Apr 11.

The absence of ER-β results in altered gene expression in ovarian granulosa cells isolated from in vivo preovulatory follicles

April K Binder  1 , Karina F Rodriguez, Katherine J Hamilton, Patricia S Stockton, Casey E Reed, Kenneth S Korach
Affiliations

The absence of ER-β results in altered gene expression in ovarian granulosa cells isolated from in vivo preovulatory follicles

April K Binder et al. Endocrinology. 2013 Jun.

Abstract

Determining the spatial and temporal expression of genes involved in the ovulatory pathway is critical for the understanding of the role of each estrogen receptor in the modulation of folliculogenesis and ovulation. Estrogen receptor (ER)-β is highly expressed in ovarian granulosa cells, and mice lacking ER-β are subfertile due to inefficient ovulation. Previous work has focused on isolated granulosa cells or cultured follicles and, although informative, provides confounding results due to the heterogeneous cell types present including granulosa and theca cells and oocytes and exposure to in vitro conditions. Herein we isolated preovulatory granulosa cells from wild-type (WT) and ERβ-null mice using laser capture microdissection to examine the genomic transcriptional response downstream of pregnant mare serum gonadotropin (mimicking FSH) and pregnant mare serum gonadotropin/human chorionic gonadotropin (mimicking LH) stimulation. This allows for a direct comparison of in vivo granulosa cells at the same stage of development from both WT and ERβ-null ovaries. ERβ-null granulosa cells showed altered expression of genes known to be regulated by FSH (Akap12 and Runx2) as well as not previously reported (Arnt2 and Pou5f1) in WT granulosa cells. Our analysis also identified 304 genes not previously associated with ERβ in granulosa cells. LH-responsive genes including Abcb1b and Fam110c show reduced expression in ERβ-null granulosa cells; however, novel genes including Rassf2 and Megf10 were also identified as being downstream of LH signaling in granulosa cells. Collectively, our data suggest that granulosa cells from ERβ-null ovaries may not be appropriately differentiated and are unable to respond properly to gonadotropin stimulation.

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Figures

Figure 1.
Figure 1.
Use of LCM to isolate preovulatory granulosa and theca cells. Immature WT and Ex3βERKO ovaries were frozen in optimum cutting temperature after exogenous gonadotropin stimulation. A, Representative ovarian section stained with 1% cresyl violet acetate prior to LCM isolation. B, Pure populations of mural granulosa cells from large preovulatory follicles were isolated. C, Theca cells were also isolated from large preovulatory follicles.
Figure 2.
Figure 2.
Confirmation of genes showing altered expression after PMSG stimulation. Granulosa cells were mechanically isolated from WT and Ex3βERKO ovaries 48 hours after PMSG stimulation. RNA was isolated and reverse transcribed, and real-time PCR was performed with primers specific for Akap12 (A), Pou5f1 (B), Arnt2 (C), Ahr (D), Trim61 (E), Lrp11 (F), Fam110c (G), Perp (H), Runx2 (I), and Reln (J). Data shown are a ratio of the gene of interest to Pl7 (used as a housekeeping gene), each ran in duplicate. Data were analyzed by an unpaired Student's t test. *, P < .05; **, P < 0,01; ***, P < .001.
Figure 3.
Figure 3.
Confirmation of genes showing altered expression after PMSG/hCG stimulation. Granulosa cells were mechanically isolated from WT and Ex3βERKO ovaries after 48 hours of PMSG and 4 hours of hCG stimulation. RNA was isolated and reverse transcribed, and real-time PCR was performed with primers specific for Hpgd (A), Abcb1b (B), Rassf2 (C), Klf4 (D), Scube1 (E), Srxn1 (F), Fam110c (G), and Megf10 (H). Data shown are a ratio of the gene of interest to Pl7 (used as a housekeeping gene), each ran in duplicate. Data were analyzed by an unpaired Student's t test. ***, P < .001.
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