Illustration of differentially expressed cell cycle genes in early CL.

<p>The diagram depicts up-regulated genes that were differentially expressed (p<0.05) in day 4 vs. 11 CL. Genes appear in the diagram in relation to cell cycle steps. Intensity of the shading increases with the magnitude of the change (see inset).</p

Clustering of bovine CL DEG on day 4 vs. day 11 using hierarchical clustering performed with Partek Genomics Suite.

<p>The Hierarchical clustering of 681 expressed genes (fold-change |≥ 2|, FDR of <5%) in day 4 and day 11 CL. Each column represents an Affymetrix chip (n = 10) and each row represents a gene. The deep red color represents relative upregulated expression, while the deep blue color represents relative down regulated expression.</p

Effect of 24h treatment with forskolin (10μM) on cholesterol biosynthetic gene expression in granulosa cells.

<p>Effect of 24h treatment with forskolin (10μM) on cholesterol biosynthetic gene expression in granulosa cells.</p

Genes, sequences and Genbank accessions of the primers used in qPCR.

<p>Genes, sequences and Genbank accessions of the primers used in qPCR.</p

Illustration of differentially expressed cholesterol biosynthetic genes in midcycle CL.

<p>The diagram shows up-regulated, DEG (p<0.05) on day 11 vs. day 4 CL. Intensity of the shading increases with the magnitude of the change (see inset).</p

Genomic profiling of bovine corpus luteum maturation

<div><p>To unveil novel global changes associated with corpus luteum (CL) maturation, we analyzed transcriptome data for the bovine CL on days 4 and 11, representing the developing vs. mature gland. Our analyses revealed 681 differentially expressed genes (363 and 318 on day 4 and 11, respectively), with ≥2 fold change and FDR of <5%. Different gene ontology (GO) categories were represented prominently in transcriptome data at these stages (e.g. days 4: cell cycle, chromosome, DNA metabolic process and replication and on day 11: immune response; lipid metabolic process and complement activation). Based on bioinformatic analyses, select genes expression in day 4 and 11 CL was validated with quantitative real-time PCR. Cell specific expression was also determined in enriched luteal endothelial and steroidogenic cells. Genes related to the angiogenic process such as <i>NOS3</i>, which maintains dilated vessels and <i>MMP9</i>, matrix degrading enzyme, were higher on day 4. Importantly, our data suggests day 11 CL acquire mechanisms to prevent blood vessel sprouting and promote their maturation by expressing <i>NOTCH4</i> and <i>JAG1</i>, greatly enriched in luteal endothelial cells. Another endothelial specific gene, <i>CD300LG</i>, was identified here in the CL for the first time. <i>CD300LG</i> is an adhesion molecule enabling lymphocyte migration, its higher levels at mid cycle are expected to support the transmigration of immune cells into the CL at this stage. Together with steroidogenic genes, most of the genes regulating de-novo cholesterol biosynthetic pathway (e.g <i>HMGCS</i>, <i>HMGCR</i>) and cholesterol uptake from plasma (<i>LDLR</i>, <i>APOD</i> and <i>APOE</i>) were upregulated in the mature CL. These findings provide new insight of the processes involved in CL maturation including blood vessel growth and stabilization, leucocyte transmigration as well as progesterone synthesis as the CL matures.</p></div

Differential expression of endothelial/blood vessel genes during CL development.

<p>mRNA levels of (A) <i>NOS3</i>, (B) <i>MMP9</i>, (C) <i>NOTCH4</i>, (D) <i>JAG1</i> and (E) <i>CD300LG</i> in the early (day 4) and mid (day 11) luteal stages. Levels of mRNA were measured by qRT-PCR and normalized to <i>RPS26</i> in the same samples. The results are presented as means ± SEM. Data were obtained from 5 cows for each luteal stage. Asterisks indicate significant differences between day 4 and day 11; *P<0.05, **P < 0.01, ***P < 0.001. The arbitrary units (Y axis) were multiplied by 100.</p

<i>STAR</i>, <i>HSD3B1</i> and <i>CYP11A1</i> expression in day 4 and day 11 CL.

<p>mRNA expression of <i>STAR</i>, <i>HSD3B1</i> and <i>CYP11A1</i> (A-C). Levels of mRNA were measured by qRT-PCR and normalized to <i>RPS26</i> in the same samples. The results are presented as means ± SEM; n = 9. Asterisks indicate significant difference between day 4 and day 11; *P<0.05, **P < 0.01, ***P < 0.001. The arbitrary units (Y axis) were multiplied by 100.</p

Luteal cell specific expression of <i>NOTCH4</i>, <i>JAG1</i>, <i>CD300LG</i> and <i>MMP9</i>.

<p>(A) <i>NOTCH4</i>, (B) <i>JAG1</i> (C) <i>CD300LG</i> and (D) <i>MMP9</i> mRNA in LSC -luteal steroidogenic cells and LEC–luteal endothelial cells enriched from mid cycle CL. Levels of mRNA were measured by qRT-PCR and normalized to <i>RPS26</i> in the same samples. The results are presented as means ± SEM; n = 9. Asterisks indicate significant differences between days 4 and 11; *P<0.05, **P < 0.01, ***P < 0.001. The arbitrary units (Y axis) were multiplied by 100.</p

Compound network representing the main underlying biological processes related to day 4 and day 11 CL.

<p>Four IPA biological networks [network 6 (score 35), network 8 (score 31), network 10 (score 28) and network 12 (score 26) see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194456#pone.0194456.s002" target="_blank">S2 File</a>] of the DEG (|fold change ≥2|, P<0.05) between day 4 and day 11 CL were merged and adopted to a larger biological network, indicating the mutual relationship between networks. The network is displayed graphically as nodes (gene/gene products) and edges (the biological relationship between nodes). Red nodes indicate genes that were upregulated in day 4 CL; green nodes indicate genes that were upregulated in day 11 CL. White nodes indicate genes that are not differentially expressed but related to this network. Intensity of the shading increases with the magnitude of the change. † Genes added manually with fold change >1.9.</p