Microarray analysis of insulin-like growth factor-I-induced changes in messenger ribonucleic acid expression in cultured porcine granulosa cells: Possible role of insulin-like growth factor-I in angiogenesis1,2

Abstract

Insulin-like growth factor-I in conjunction with gonadotropins are important stimulators of mitosis and ovarian steroid production by granulosa and thecal cells, which are required for normal oocyte development and hormonal feedback signaling to the hypothalamus and pituitary. However, a comprehensive evaluation of the changes in gene expression induced by IGF-I has not been conducted. Our objective was to characterize granulosa cell gene expression in response to IGF-I treatment. Porcine granulosa cells were pooled in 4 biological replicates and treated with FSH (baseline) or FSH+IGF-I for 24 h in vitro. The RNA was collected and hybridized to 8 Affymetrix Porcine GeneChips (Affymetrix, Santa Clara, CA) in a paired design. Differentially regulated gene sequence element sets (P < 0.01) were used as queries in the UniGene database searching for annotated genes. Abundance of messenger RNA (mRNA) for genes differentially expressed in the microarray analysis was determined through multiplex assays of one-step real-time reverse transcription-PCR and further analyzed under a statistical model including the fixed effect of treatment. A total of 388 gene sequence element sets were differentially expressed, and 42 matched annotated genes in the UniGene database. Of the 3 upregulated target genes selected for further quantitative reverse transcription-PCR analysis, only FGF receptor 2 III c (FGFR2IIIc) mRNA abundance was significantly increased by IGF-I. Of the 3 downregulated target genes selected for further analysis, only thrombospondin-1 (THBS1) mRNA abundance was significantly decreased by IGF-I. Further study revealed that neither FSH nor estradiol affected the IGF-I-induced suppression of THBS1 mRNA abundance. These results provide the first comprehensive assessment of IGF-I-induced gene expression in granulosa cells and will contribute to a better understanding of the molecular mechanisms of IGF-I regulation of follicular development. Involvement of FGFR2IIIc and THBS1 in mediating IGF-I-induced granulosa cell steroidogenesis and proliferation during follicular development is novel, but their specific roles will require further elucidation.

Rapid Insulin-like Growth Factor-1-induced Changes in Granulosa Cell Thrombospondin-1 Expression In Vitro

“…As TSP-1 is known to be a potent inhibitor of angiogenesis (reviewed in [24]), the studies presented here provide further evidence for the pro-angiogenic influence of IGF-1 in the ovary and suggest that a reduction in TSP-1 expression occurs to allow a rapid shift towards a pro-angiogenesic environment at a key stage in the developing follicle. Very recent studies in the porcine follicle demonstrate that IGF-1 significantly suppresses TSP-1 mRNA expression in granulosa cells, strongly supporting the results described here [8]. These studies extend the mRNA findings by demonstrating a mechanism behind the rapid decay and confirming that the change in mRNA level is rapidly mirrored in the expression of TSP-1 protein.…”

“…In very recent studies with primary cultures of porcine granulosa cells, it has been demonstrated that multiple mRNA species are positively and negatively regulated by IGF-1, further supporting the potential importance of this growth factor in granulosa cell function [8]. Among the genes suppressed by IGF-1 is thrombospondin-1 (TSP-1) [8].Thrombospondin-1 (TSP-1), a homotrimeric glycoprotein that participates in a wide range of biological processes, including cell proliferation, differentiation, cell adhesion, angiogenesis, cell-cell and cell-matrix interactions [9][10][11]. We have previously demonstrated that the expression of TSP-1 and TSP-2 during the bovine ovarian cycle is gonadotrophin-dependent and have identified distinct patterns of TSP-1 expression corresponding to different follicular stages [12].…”

“…IGF-1 promotes both proliferation and differentiation of granulosa cells [5], increases estradiol secretion by granulosa cells [6], and sensitizes granulosa cells to FSH signalling during late-stage follicular growth by increasing FSH receptor concentrations on granulosa cell surfaces [7]. In very recent studies with primary cultures of porcine granulosa cells, it has been demonstrated that multiple mRNA species are positively and negatively regulated by IGF-1, further supporting the potential importance of this growth factor in granulosa cell function [8]. Among the genes suppressed by IGF-1 is thrombospondin-1 (TSP-1) [8].…”

“…In very recent studies with primary cultures of porcine granulosa cells, it has been demonstrated that multiple mRNA species are positively and negatively regulated by IGF-1, further supporting the potential importance of this growth factor in granulosa cell function [8]. Among the genes suppressed by IGF-1 is thrombospondin-1 (TSP-1) [8].…”

“…Other than gonadotrophins, factors that regulate granulosa cell TSP-1 gene expression, and the mechanisms through which they mediate such changes, have not been widely studied, even though regulation by local factors such as IGF-1 is important in other contexts [5,6]. One very recent study [8] has demonstrated that TSP-1 is among several transcripts regulated by IGF-1 in primary cultures of porcine granulosa cells. However, neither the mechanism behind the decrease in TSP-1 Inducible changes in the transcription of mRNAs encoded by specific genes are key mechanisms in the cellular regulation of gene expression.…”