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

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)-? is highly expressed in ovarian granulosa cells, and mice lacking ER-? 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 ER?-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 ER?-null ovaries. ER?-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 ER? in granulosa cells. LH-responsive genes including Abcb1b and Fam110c show reduced expression in ER?-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 ER?-null ovaries may not be appropriately differentiated and are unable to respond properly to gonadotropin stimulation.

SUBMITTER: Binder AK

PROVIDER: S-EPMC3740481 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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

Binder April K AK Rodriguez Karina F KF Hamilton Katherine J KJ Stockton Patricia S PS Reed Casey E CE Korach Kenneth S KS

Endocrinology 20130411 6

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 h ...[more]

PMID: 23580569

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Project description:The postpartum period in dairy cows is associated with a state of temporary negative energy balance and could induce functional changes into ovarian granulosa cells (GC) resulting in significant impact on the ovarian function and fertility. Yet, the regulation of interleukin receptors (ILRs) in GC as well as ILs expression profile during the postpartum period have not been fully investigated. We hypothesized that the postpartum period is associated with changes in ILs expression profile that could affect follicular development and ovulation rate. First, we aimed to investigate the expression and regulation of different IL and IL receptors in GC at different stages of follicular development and then analyse the changes in target ILs expression profile induced during the postpartum period. In the first objective, normal cycling cows were selected and GC were collected from small follicles (SF), dominant follicles at day 5 of the estrous cycle (DF), and ovulatory follicles, 24 h following hCG injection (OF). In the second objective, dairy cows between 50 and 70 days postpartum were randomly selected, and ?-hydroxybutyrate (BHB) concentrations were measured in blood samples in order to assign cows to the BHB+ group (>1.4 mmol/L) or BHB- group (<1.2 mmol/L). GC were collected from preovulatory follicles by transvaginal aspiration. Total RNA was extracted from GC of all groups for analysis of target ILs and ILRs expression. Steady-state mRNA levels of IL4R was strongest in the DF, while IL15R expression was greatest in the OF, and IL21R showed increased steady-state mRNA levels in the corpus luteum as compared to the different groups of follicles. Overall, expression of IL1A, IL1B, IL8, IL15, IL23 and TNF? was stronger in OF as compared to DF, while IL4 and IL10 expression was stronger in SF than in DF. Similarly, expression of IL1A, IL1B, IL8, IL15, IL23, and TNF? were significantly stronger in GC of BHB+ cows than in the control, while IL4 expression was significantly reduced in BHB+ as compared to control cows. We have established an IL expression profile, which suggest a correlation with BHB levels during the postpartum period. Additionally, we have demonstrated a differential regulation of target ILRs in GC at different stages of follicular development. Overall, these data provide a better understanding of the changes that could affect follicular development and ovulation during the postpartum period and lay the ground for further investigations.

Project description:BackgroundThe major function of the ovary is to produce oocytes for fertilisation. Oocytes mature in follicles surrounded by nurturing granulosa cells and all are enclosed by a basal lamina. During growth, granulosa cells replicate and a large fluid-filled cavity (the antrum) develops in the centre. Only follicles that have enlarged to over 10 mm can ovulate in cows. In mammals, the number of primordial follicles far exceeds the numbers that ever ovulate and atresia or regression of follicles is a mechanism to regulate the number of oocytes ovulated and to contribute to the timing of ovulation. To better understand the molecular basis of follicular atresia, we undertook transcriptome profiling of granulosa cells from healthy (n = 10) and atretic (n = 5) bovine follicles at early antral stages (< 5 mm).ResultsPrincipal Component Analysis (PCA) and hierarchical classification of the signal intensity plots for the arrays showed primary clustering into two groups, healthy and atretic. These analyses and size-frequency plots of coefficients of variation of signal intensities revealed that the healthy follicles were more heterogeneous. Examining the differentially-expressed genes the most significantly affected functions in atretic follicles were cell death, organ development, tissue development and embryonic development. The overall processes influenced by transcription factor gene TP53 were predicted to be activated, whereas those of MYC were inhibited on the basis of known interactions with the genes in our dataset. The top ranked canonical pathway contained signalling molecules common to various inflammatory/fibrotic pathways such as the transforming growth factor-β and tumour necrosis factor-α pathways. The two most significant networks also reflect this pattern of tissue remodelling/fibrosis gene expression. These networks also contain molecules which are present in the canonical pathways of hepatic fibrosis/hepatic stellate cell activation and transforming growth factor-β signalling and were up regulated.ConclusionsSmall healthy antral follicles, which have a number of growth outcomes, exhibit greater variability in gene expression, particularly in genes associated with cell division and other growth-related functions. Atresia, on the other hand, not only involves cell death but clearly is an active process similar to wound healing.

Dataset Information

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

Publications

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

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