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Hormonal regulation of MicroRNA expression in periovulatory mouse mural granulosa cells.

ABSTRACT: MicroRNAs (miRNAs) mediate posttranscriptional gene regulation by binding to the 3' untranslated region of messenger RNAs to either inhibit or enhance translation. The extent and hormonal regulation of miRNA expression by ovarian granulosa cells and their role in ovulation and luteinization is unknown. In the present study, miRNA array analysis was used to identify 212 mature miRNAs as expressed and 13 as differentially expressed in periovulatory granulosa cells collected before and after an ovulatory dose of hCG. Two miRNAs, Mirn132 and Mirn212 (also known as miR-132 and miR-212), were found to be highly upregulated following LH/hCG induction and were further analyzed. In vivo and in vitro temporal expression analysis by quantitative RT-PCR confirmed that LH/hCG and cAMP, respectively, increased transcription of the precursor transcript as well as the mature miRNAs. Locked nucleic acid oligonucleotides complementary to Mirn132 and Mirn212 were shown to block cAMP-mediated mature miRNA expression and function. Computational analyses indicated that 77 putative mRNA targets of Mirn132 and Mirn212 were expressed in ovarian granulosa cells. Furthermore, upon knockdown of Mirn132 and Mirn212, a known target of Mirn132, C-terminal binding protein 1, showed decreased protein levels but no change in mRNA levels. The following studies are the first to describe the extent of miRNA expression within ovarian granulosa cells and the first to demonstrate that LH/hCG regulates the expression of select miRNAs, which affect posttranscriptional gene regulation within these cells.

SUBMITTER: Fiedler SD

PROVIDER: S-EPMC2780477 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Hormonal regulation of MicroRNA expression in periovulatory mouse mural granulosa cells.

Fiedler Stephanie D SD Carletti Martha Z MZ Hong Xiaoman X Christenson Lane K LK

Biology of reproduction 20080820 6

MicroRNAs (miRNAs) mediate posttranscriptional gene regulation by binding to the 3' untranslated region of messenger RNAs to either inhibit or enhance translation. The extent and hormonal regulation of miRNA expression by ovarian granulosa cells and their role in ovulation and luteinization is unknown. In the present study, miRNA array analysis was used to identify 212 mature miRNAs as expressed and 13 as differentially expressed in periovulatory granulosa cells collected before and after an ovu ...[more]

PMID: 18716288

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Project description:BACKGROUND: Ovarian follicle development is a complex process. Paracrine interactions between somatic and germ cells are critical for normal follicular development and oocyte maturation. Studies have suggested that the health and function of the granulosa and cumulus cells may be reflective of the health status of the enclosed oocyte. The objective of the present study is to assess, using an in vivo immature rat model, gene expression profile in granulosa cells, which may be linked to the developmental competence of the oocyte. We hypothesized that expression of specific genes in granulosa cells may be correlated with the developmental competence of the oocyte. METHODS: Immature rats were injected with eCG and 24 h thereafter with anti-eCG antibody to induce follicular atresia or with pre-immune serum to stimulate follicle development. A high percentage (30-50%, normal developmental competence, NDC) of oocytes from eCG/pre-immune serum group developed to term after embryo transfer compared to those from eCG/anti-eCG (0%, poor developmental competence, PDC). Gene expression profiles of mural granulosa cells from the above oocyte-collected follicles were assessed by Affymetrix rat whole genome array. RESULTS: The result showed that twelve genes were up-regulated, while one gene was down-regulated more than 1.5 folds in the NDC group compared with those in the PDC group. Gene ontology classification showed that the up-regulated genes included lysyl oxidase (Lox) and nerve growth factor receptor associated protein 1 (Ngfrap1), which are important in the regulation of protein-lysine 6-oxidase activity, and in apoptosis induction, respectively. The down-regulated genes included glycoprotein-4-beta galactosyltransferase 2 (Ggbt2), which is involved in the regulation of extracellular matrix organization and biogenesis. CONCLUSIONS: The data in the present study demonstrate a close association between specific gene expression in mural granulosa cells and the developmental competence of oocytes. This finding suggests that the most differentially expressed gene, lysyl oxidase, may be a candidate biomarker of oocyte health and useful for the selection of good quality oocytes for assisted reproduction.

Project description:Vascular endothelial growth factor A (VEGFA) stimulates angiogenesis and is associated with increased vascularity in ovarian follicles of cattle. The objectives of this study were to investigate the developmental and hormonal regulation of VEGFA expression in ovarian granulosa and theca cells (TC) of cattle. Bovine ovaries were collected from a local slaughterhouse and granulosa cells (GC) and TC were collected from small (SM; 1 to 5 mm) and large (LG; 8 to 20 mm) follicles. Cells were collected fresh or cultured in serum-free medium and treated with various factors that regulate angiogenesis and follicular development. RNA was collected for analysis of VEGFA mRNA abundance via quantitative PCR. In SM-follicle GC (SMGC), prostaglandin E2 (PGE2) and FSH decreased (P < 0.05) VEGFA mRNA abundance by 30 to 46%, whereas in LG-follicle GC (LGGC), PGE2 and FSH were without effect (P > 0.10). In SMGC, dihydrotestosterone (DHT), sonic hedgehog (SHH), and growth differentiation factor-9 (GDF9) decreased (P < 0.05) VEGFA expression by 30 to 40%. Fibroblast growth factor-9 (FGF9) and estradiol (E2) were without effect (P > 0.10) on VEGFA mRNA in both SMGC and LGGC, whereas progesterone increased (P < 0.05) VEGFA mRNA in LGGC but had no effect in LGTC. Bone morphogenetic protein-4 (BMP4), LH, and FGF9 increased (P < 0.05) abundance of VEGFA mRNA by 1.5- to 1.9-fold in LGTC. Insulin-like growth factor-1 (IGF1) was without effect (P > 0.10) on VEGFA mRNA in both TC and GC. An E2F transcription factor inhibitor, HLM0064741 (E2Fi), dramatically (i.e., 8- to 13-fold) stimulated (P < 0.01) the expression of VEGFA mRNA expression in both SMGC and LGTC. Abundance of VEGFA mRNA was greater (P < 0.05) in LGGC and SMGC than in LGTC. Also, SMTC had greater (P < 0.05) abundance of VEGFA mRNA than LGTC. In conclusion, VEGFA mRNA abundance was greater in GC than TC, and VEGFA expression decreased in TC during follicle development. Some treatments either suppressed, stimulated, or had no effect on VEGFA expression depending on the cell type. The inhibition of E2F transcription factors had the greatest stimulatory effect of all treatments evaluated, and thus, E2Fs may play an important role in regulating angiogenesis during follicle growth in cattle.

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Hormonal regulation of MicroRNA expression in periovulatory mouse mural granulosa cells.

Publications

Hormonal regulation of MicroRNA expression in periovulatory mouse mural granulosa cells.

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