Induction of altered gene expression profiles in cultured bovine granulosa cells at high cell density

Abstract

BackgroundIn previous studies it has been shown that bovine granulosa cells (GC) cultured at a high plating density dramatically change their physiological and molecular characteristics, thus resembling an early stage of luteinization. During the present study, these specific effects on the GC transcriptome were comprehensively analysed to clarify the underlying mechanisms.MethodsGC were cultured in serum free medium with FSH and IGF-1 stimulation at different initial plating density. The estradiol and progesterone production was determined by radioimmunoassays and the gene expression profiles were analysed by mRNA microarray analysis after 9 days. The data were statistically analysed and the abundance of selected, differentially expressed transcripts was re-evaluated by qPCR. Bioinformatic pathway analysis of density affected transcripts was done using Ingenuity Pathway Analysis.ResultsThe data showed that at high plating density the expression of 1510 annotated genes, represented by 1575 transcript clusters, showed highly altered expression levels. Nearly two-thirds were up- and one third down-regulated. Within the top up-regulated genes VNN2, RGS2 and PTX3 could be identified, as well as HBA or LOXL2. Down-regulated genes included important key genes of folliculogenesis like CYP19A1 and FSHR. Ingenuity pathway analysis identified “AMPK signaling” as well as “cAMP-mediated signaling” as major pathways affected by the alteration of the expression profile. Main putative upstream regulators were TGFB1 and VEGF, thus indicating a connection with cell differentiation and angiogenesis. A detailed cluster analysis revealed one single cluster that was highly associated with the upstream regulator beta-estradiol. Within this cluster key genes of steroid biosynthesis were not included, but instead, other genes importantly involved in follicular development, like OXT and VEGFA as well as the three most down-regulated genes TXNIP, PAG11 and ARRDC4 were identified.ConclusionsFrom these data we hypothesize that high density conditions induce a stage of differentiation in cultured GC that is similar to early post-LH conditions in vivo. Furthermore we hypothesize that specific cell-cell-interactions led to this differentiation including transformations necessary to promote angiogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-016-0221-6) contains supplementary material, which is available to authorized users.

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

“…p > 0.05, two-tailed Student's t -test. This figure is reproduced from Baufeld et al 15 . Please click here to view a larger version of this figure.…”

“…To determine the transcript abundance, synthesize cDNA and measure specific transcripts by quantitative real-time polymerase chain reaction (qPCR) techniques 15 . A statistical evaluation should include at least three replications of different cell cultures, originated from different cell preparations (biological replicates).…”

Lactate promotes specific differentiation in bovine granulosa cells depending on lactate uptake thus mimicking an early post-LH stage

“…In ovarian tissue and granulosa cells expression of specific subunits of the NMDA receptor, the subunits 1 and 2B, had been identified [ 50 ] thus supporting the view that this receptor might be involved in these cells as well. Expression of NMDAR subunits, especially of GRIN2D and GRINA has been found in vivo in GC (see database GranulosaIMAGE [ 51 ]) as well as in vitro (own GC culture microarray dataset [ 14 ]). Whether these receptor subunits can actually form functional receptors to be modulated by L-lactate in bovine GC has to be investigated in further studies.…”

“…Likewise, we showed that high cell density induces specific changes of the gene expression and steroid hormone profiles in cultured GC mimicking an early post-LH status of GC differentiation [ 13 ]. Furthermore, applying a whole-genome approach we could demonstrate in GC cultured at high plating density, that genes connected to hypoxia are affected [ 14 ]. In particular LDHA transcripts encoding L-lactate dehydrogenase were remarkably up-regulated.…”

HuoXueJieDu Formula Alleviates Diabetic Retinopathy in Rats by Inhibiting SOCS3-STAT3 and TIMP1-A2M Pathways

“…Subsequently, pathway enrichment analysis was performed using the Ingenuity Pathway Analysis (IPA) tool. P values were calculated using Fisher's exact test, and P values <0.05 were considered significantly enriched in the canonical pathways [ 11 ].…”